Apolipoprotein E receptors mediate the effects of beta-amyloid on astrocyte cultures

We have previously shown that beta-amyloid (Abeta) induces astrocyte activation in vitro and that this reaction is attenuated by the addition of exogenous apolipoprotein E (apoE)-containing particles. However, the effects of Abeta on endogenous apoE and apoJ levels and the potential role of apoE receptors in astrocyte activation have not been addressed. Three activating stimuli (lipopolysaccharide, dibutyryl cAMP, and aged Abeta 1-42) were used to induce activation of rat astrocyte cultures, as assessed by changes in morphology and an increase in interleukin-1beta. However, only Abeta also induced approximately 50% reduction in the amount of released apoE and apoJ and an 8-fold increase in the levels of cell-associated apoE and apoJ. Experiments using two concentrations of receptor-associated protein, an inhibitor of apoE receptors with a differential affinity for the low density lipoprotein receptor (LDLR) and the LDLR-related protein (LRP), suggest that LRP mediates Abeta-induced astrocyte activation, whereas LDLR mediates the Abeta-induced changes in apoE levels. Receptor-associated protein had no effect on apoJ levels or on activation by either dibutyryl cAMP or lipopolysaccharide. These data suggest that apoE receptors translate the presence of extracellular Abeta into cellular responses, both initiating and modulating the inflammatory response induced by Abeta.     

Analysis of expression of genes involved in apolipoprotein E-based lipoprotein metabolism in pregnant mice deficient in the receptor-associated protein, the low density lipoprotein receptor, or apolipoprotein E.

Mice deficient in receptor-associated protein (RAP) were phenotypically normal, but in contrast to results previously reported in RAP(-/-) mice, nearly 50% of the offspring died at or shortly after birth. To attempt to determine the reason for this, we analyzed the regulation of expression of genes involved in apolipoprotein E (apoE)-based mechanisms in RAP-deficient mice and compared this to results in mice deficient in low density lipoprotein receptor (LDLR) or apoE. The major finding concerned a large increase in hepatic lipoprotein receptor-related protein (LRP) mRNA and LDLR mRNA levels in pregnant RAP knockout mice. This is in contrast to the down-regulation of LRP mRNA and LDLR mRNA, which is normally seen in wild-type mice. Also in LDLR knockout mice, a significant up-regulation in expression of LRP mRNA was demonstrated. In apoE knockout mice, hepatic LRP mRNA did not change significantly, while hepatic LDLR mRNA expression was increased. In placenta and uterus, the deficiency of RAP did not markedly affect the expression of LRP and LDLR. Lipoprotein lipase mRNA and apoE mRNA increased during pregnancy in all mice, independent of their genetic status. The current study does not directly explain the increased mortality of RAP(-/-) pups. The data demonstrate, however, important relative changes in expression of the genes analyzed, an indication that LRP and LDLR play an important role in lipid metabolism during pregnancy.     

Receptor-associated Protein Interacts with Amyloid-�� Peptide and Promotes Its Cellular Uptake

Brain amyloid-β (Aβ) peptide accumulation and aggregation are critical events in the pathogenesis of Alzheimer disease. Increasing evidence has demonstrated that LRP1 is involved in Alzheimer disease pathogenesis. The physiological ligands of LRP1, including apoE, play significant roles in the cellular clearance of Aβ. The receptor-associated protein (RAP) is a specialized chaperone for members of the low density lipoprotein receptor family. RAP shares structural and receptor-binding properties with apoE. Here, we show that RAP binds to both Aβ40 and Aβ42 in a concentration-dependent manner and forms complexes with them. Fluorescence-activated cell sorter analysis showed that RAP significantly enhances the cellular internalization of Aβ in different cell types, including brain vascular smooth muscle, neuroblastoma, glioblastoma, and Chinese hamster ovary cells. This effect of RAP was confirmed by fluorescence microscopy and enzyme-linked immunosorbent assay. RAP binds to both LRP1 and heparin; however, the ability of RAP to enhance Aβ cellular uptake was blocked by heparin and heparinase treatment but not by LRP1 deficiency. Furthermore, the effects of RAP were significantly decreased in heparan sulfate proteoglycan-deficient Chinese hamster ovary cells. Our findings reveal that RAP is a novel Aβ-binding protein that promotes cellular internalization of Aβ.     

An ABCA1-independent pathway for recycling a poorly lipidated 8.1 nm apolipoprotein E particle from glia

Lipid transport in the brain is coordinated by glial-derived lipoproteins that contain apolipoprotein E (apoE) as their primary protein. Here we show that apoE is secreted from wild-type (WT) primary murine mixed glia as nascent lipoprotein subspecies ranging from 7.5 to 17 nm in diameter. Negative-staining electron microscropy (EM) revealed rouleaux, suggesting a discoidal structure. Potassium bromide (KBr) density gradient ultracentrifugation showed that all subspecies, except an 8.1 nm particle, were lipidated. Glia lacking the cholesterol transporter ABCA1 secreted only 8.1 nm particles, which were poorly lipidated and nondiscoidal but could accept lipids to form the full repertoire of WT apoE particles. Receptor-associated-protein (RAP)-mediated inhibition of apoE receptor function blocked appearance of the 8.1 nm species, suggesting that this particle may arise through apoE recycling. Selective deletion of the LDL receptor (LDLR) reduced the level of 8.1 nm particle production by approximately 90%, suggesting that apoE is preferentially recycled through the LDLR. Finally, apoA-I stimulated secretion of 8.1 nm particles in a dose-dependent manner. These results suggest that nascent glial apoE lipoproteins are secreted through multiple pathways and that a greater understanding of these mechanisms may be relevant to several neurological disorders.     

Apolipoprotein E Attenuates β-Amyloid-Induced Astrocyte Activation

Abstract: A common feature of Alzheimer's disease pathology is an abundance of activated glia, indicative of an inflammatory reaction in the brain. The relationship between glial activation and neurodegeneration is not known, although several cytokines and inflammatory mediators produced by activated glia have the potential to initiate or exacerbate the progression of neuropathology. As β-amyloid (Aβ) is one of several stimuli that can activate glia, it is important to determine how Aβ-induced glial activation is influenced by other proteins present in the plaque, such as apolipoprotein E (apoE). We examined the effect of native preparations of apoE on activation of rat cortical astrocyte cultures by Aβ1–42. The apoE source was conditioned medium from human embryonic kidney 293 cells stably transfected with human apoE3 or apoE4 cDNA. By morphological criteria, apoE inhibited Aβ-induced astrocyte activation in three experimental paradigms: apoE pretreatment blocked subsequent Aβ-induced activation, Aβ aged in the presence of apoE did not activate astrocytes, and apoE addition to activated astrocytes transiently reversed the activated phenotype. No apoE isoform selectivity was observed. The effect of apoE appears to be specific to Aβ, as apoE did not attenuate cyclic AMP-induced astrocyte activation. These data suggest that apoE may modulate the ability of Aβ to induce inflammatory responses in the brain.     

Molecular chaperone RAP interacts with LRP1 in a dynamic bivalent mode and enhances folding of ligand-binding regions of other LDLR family receptors

The low-density lipoprotein receptor (LDLR) family of receptors are cell-surface receptors that internalize numerous ligands and play crucial role in various processes, such as lipoprotein metabolism, hemostasis, fetal development, etc. Previously, receptor-associated protein (RAP) was described as a molecular chaperone for LDLR-related protein 1 (LRP1), a prominent member of the LDLR family. We aimed to verify this role of RAP for LRP1 and two other LDLR family receptors, LDLR and vLDLR, and to investigate the mechanisms of respective interactions using a cell culture model system, purified system, and in silico modelling. Upon coexpression of RAP with clusters of the ligand-binding complement repeats (CRs) of the receptors in secreted form in insect cells culture, the isolated proteins had increased yield, enhanced folding, and improved binding properties compared with proteins expressed without RAP, as determined by circular dichroism and surface plasmon resonance. Within LRP1 CR-clusters II and IV, we identified multiple sites comprised of adjacent CR doublets, which provide alternative bivalent binding combinations with specific pairs of lysines on RAP. Mutational analysis of these lysines within each of isolated RAP D1/D2 and D3 domains having high affinity to LRP1 and of conserved tryptophans on selected CR-doublets of LRP1, as well as in silico docking of a model LRP1 CR-triplet with RAP, indicated a universal role for these residues in interaction of RAP and LRP1. Consequently, we propose a new model of RAP interaction with LDLR family receptors based on switching of the bivalent contacts between molecules over time in a dynamic mode.     

Chylomicron remnant uptake in the livers of mice expressing human apolipoproteins E3, E2 (Arg158-->Cys), and E3-Leiden

Apolipoprotein E2 (apoE2) and apoE3-Leiden cause chylomicron remnant accumulation (type III hyperlipidemia). However, the degree of dyslipidemia and its penetrance are different in humans and mice. Remnant uptake by isolated liver from apoE-/- mice transgenic for human apoE2, apoE3-Leiden, or apoE3 was measured. In the presence of both LDL receptor (LDLR) and LDL receptor-related protein (LRP), remnant uptake was apoE3>E3-Leiden>E2 mice. Absence of LDLR reduced uptake in apoE3 and apoE3-Leiden-secreting livers but not in apoE2-secreting livers. LRP inhibition with receptor-associated protein reduced uptake in apoE3- and apoE2-secreting livers, but not in apoE3-Leiden-secreting livers, regardless of the presence of LDLR. Fluorescently labeled remnants clustered with LRP in apoE3-secreting livers only in the absence of LDLR, but clustered in livers that expressed apoE2 even in the presence of LDLR, and did not cluster with LRP in livers of apoE3-Leiden even in the absence of LDLR. Remnants were reconstituted with the three human apoE isoforms. Removal by liver of mApoe-/-/mldlr-/- mice expressing the human LDLR was slightly greater than removal in the previous experiments with apoE3>E2> E3-Leiden. Thus, in vivo, human apoE2 is cleared primarily by LRP, apoE3-Leiden is cleared only by the LDLR, and apoE3 is cleared by both.     

Low density lipoprotein receptor-related protein mediates apolipoprotein E inhibition of smooth muscle cell migration.

This research was undertaken to identify the cell surface receptor responsible for mediating apolipoprotein E (apoE) inhibition of platelet-derived growth factor (PDGF)-directed smooth muscle cell migration. Initial studies revealed the expression of the low density lipoprotein receptor (LDLR), the LDL receptor-related protein (LRP), the very low density lipoprotein receptor (VLDL), and apoE receptor-2 in mouse aortic smooth muscle cells. Smooth muscle cells isolated from LDLR-null, VLDL-null, and apoE receptor-2-null mice were responsive to apoE inhibition of PDGF-directed smooth muscle cell migration, suggesting that these receptors were not involved. An antisense RNA expression knockdown strategy, utilizing morpholino antisense RNA against LRP, was used to reduce LRP expression in smooth muscle cells to assess the role of this receptor in apoE inhibition of cell migration. Results showed that apoE was unable to inhibit PDGF-directed migration of LRP-deficient smooth muscle cells. The role of LRP in mediating apoE inhibition of PDGF-directed smooth muscle cell migration was confirmed by experiments showing that antibodies against LRP effectively suppressed apoE inhibition of PDGF-directed smooth muscle cell migration. Taken together, these results document that apoE binding to LRP is required for its inhibition of PDGF-directed smooth muscle cell migration.     

Apolipoprotein E Structure and Substrate and Receptor-Binding Activities of Triglyceride-Rich Human Plasma Lipoproteins in Normo- and Hypertriglyceridemia

Cysteine-arginine interchanges along the primary sequence of human plasma apolipoprotein E (apoE) play an important role in determining its biological functions due to a high mutation frequency of cytosine in CGX triplet that codes 33 of 34 apolipoprotein arginine residues. The contribution of apoE secondary structure to apolipoprotein-lipid interaction is described. The significance of apolipoprotein in triglyceride synthesis, lipoprotein lipolysis, and receptor-mediated clearance of lipolytic remnants of triglyceride-rich lipoproteins is discussed as well. The metabolic flow of lipoproteins in normo- and hypertriglyceridemia can be described by separate compartments that contribute to lipoprotein interaction with at least six different receptors: 1) low density lipoprotein (LDL) receptor; 2) LDL receptor-related protein (LRP); 3) apoB(48) macrophage receptor for hypertriglyceridemic very low density lipoproteins (VLDL); 4) scavenger receptors; 5) VLDL receptor; 6) lipolysis-stimulated receptor. The contribution of the exposure of apoE molecules on the surface of triglyceride-rich particles sensitive both to lipolysis and plasma triglyceride content to the interaction with LDL receptor and LRP is emphasized.     

Low density lipoprotein receptor-related protein 1 dependent endosomal trapping and recycling of apolipoprotein E

Background

Lipoprotein receptors from the low density lipoprotein (LDL) receptor family are multifunctional membrane proteins which can efficiently mediate endocytosis and thereby facilitate lipoprotein clearance from the plasma. The biggest member of this family, the LDL receptor-related protein 1 (LRP1), facilitates the hepatic uptake of triglyceride-rich lipoproteins (TRL) via interaction with apolipoprotein E (apoE). In contrast to the classical LDL degradation pathway, TRL disintegrate in peripheral endosomes, and core lipids and apoB are targeted along the endocytic pathway for lysosomal degradation. Notably, TRL-derived apoE remains within recycling endosomes and is then mobilized by high density lipoproteins (HDL) for re-secretion. The aim of this study is to investigate the involvement of LRP1 in the regulation of apoE recycling.

Principal Findings

Immunofluorescence studies indicate the LRP1-dependent trapping of apoE in EEA1-positive endosomes in human hepatoma cells. This processing is distinct from other LRP1 ligands such as RAP which is efficiently targeted to lysosomal compartments. Upon stimulation of HDL-induced recycling, apoE is released from LRP1-positive endosomes but is targeted to another, distinct population of early endosomes that contain HDL, but not LRP1. For subsequent analysis of the recycling capacity, we expressed the full-length human LRP1 and used an RNA interference approach to manipulate the expression levels of LRP1. In support of LRP1 determining the intracellular fate of apoE, overexpression of LRP1 significantly stimulated HDL-induced apoE recycling. Vice versa LRP1 knockdown in HEK293 cells and primary hepatocytes strongly reduced the efficiency of HDL to stimulate apoE secretion.

Conclusion

We conclude that LRP1 enables apoE to accumulate in an early endosomal recycling compartment that serves as a pool for the intracellular formation and subsequent re-secretion of apoE-enriched HDL particles.     

Cellular catabolism of lipid poor apolipoprotein E via cell surface LDL receptor-related protein

Apolipoprotein E (apoE), an apoprotein involved in lipid transport in both the plasma and within the brain, mediates the binding of lipoproteins to members of the low density lipoprotein (LDL) receptor family including the LDL receptor and the LDL receptor-related protein (LRP). ApoE/LRP interactions may be particularly important in brain where both are expressed at high levels, and polymorphisms in the apoE and LRP genes have been linked to AD. To date, only apoE-enriched lipoproteins have been shown to be LRP ligands. To investigate further whether other, more lipid-poor forms of apoE interact with LRP, we tested whether lipid-free apoE in the absence of lipoprotein particles interacts with its cell-surface receptors. No detectable lipid was found associated with bacterially expressed and purified apoE either prior to or following incubation with cells when analyzed by electrospray ionization mass spectrometry. We found that the degradation of lipid-poor (125)I-apoE was significantly higher in wild type as compared to LRP-deficient cells, and was inhibited by receptor-associated protein (RAP). In contrast, (125)I-apoE-enriched beta-VLDL was degraded by both LRP and the LDL receptor. When analyzed via a single cycle of endocytosis, (125)I-apoE was internalized prior to its subsequent intracellular degradation with kinetics typical of receptor-mediated endocytosis. Thus, we conclude that a very lipid-poor form of apoE can be catabolized via cell surface LRP, suggesting that the conformation of apoE necessary for recognition by LRP can be imposed by situations other than an apoE-enriched lipoprotein.     

RAP,a specialized chaperone,prevents ligand-induced ER retention and degradation of LDL receptor-related endocytic receptors.

The multifunctional low density lipoprotein (LDL) receptor-related protein (LRP) forms a complex with a receptor-associated protein (RAP) within the secretory pathway. RAP inhibits ligand binding to LRP and is required for normal functional expression of LRP in vivo, suggesting a physiological function as a specialized chaperone. We have used RAP-deficient mice, generated by gene targeting, to investigate the role of RAP in the biosynthesis and biological activity of LRP and other members of the LDL receptor gene family in various organs and in embryonic fibroblasts. Our results demonstrate that RAP is required for the proper folding and export of the receptors from the endoplasmic reticulum (ER) by preventing the premature binding of co-expressed ligands. Overexpression of apolipoprotein E (apoE), a high affinity ligand for LRP, results in dramatically reduced cellular LRP expression, an effect that is prevented by co-expression of RAP. RAP thus defines a novel class of molecular chaperones that selectively protect endocytic receptors by binding to newly synthesized receptor polypeptides, thereby preventing ligand-induced aggregation and subsequent degradation in the ER.     

Estrogen (E2) and glucocorticoid (Gc) effects on microglia and A beta clearance in vitro and in vivo

The accumulation of fibrillar aggregates of beta Amyloid (Aβ) in Alzheimer's Disease (AD) brain is associated with chronic brain inflammation. Although activated microglia (μglia) can potentially clear toxic amyloid, chronic activation may lead to excessive production of neurotoxins. Recent epidemiological and clinical data have raised questions about the use of anti-inflammatory steroids (glucocorticoids, Gcs) and estrogens for treatment or prevention of AD. Since very little is known about steroid effects on μglial interactions with amyloid, we investigated the effects of the synthetic Gc dexamethasone (DXM) and 17-β estradiol (E2) in vitro in a murine μglial-like N9 cell line on toxin production and intracellular Aβ accumulation. To determine whether the steroid alterations of Aβ uptake in vitro had relevance in vivo, we examined the effects of these steroids on Aβ accumulation and μglial responses to Aβ infused into rat brain. Our in vitro data demonstrate for the first time that Gc dose-dependently enhanced μglial Aβ accumulation and support previous work showing that E2 enhances Aβ uptake. Despite both steroids enhancing uptake, degradation was impeded, particularly with Gcs. Distinct differences between the two steroids were observed in their effect on toxin production and cell viability. Gc dose-dependently increased toxicity and potentiated Aβ induction of nitric oxide, while E2 promoted cell viability and inhibited Aβ induction of nitric oxide. The steroid enhancement of μglial uptake and impedence of degradation observed in vitro were consistent with observations from in vivo studies. In the brains of Aβ-infused rats, the μglial staining in entorhinal cortex layer 3, not associated with Aβ deposits was increased in response to Aβ infusion and this effect was blocked by feeding rats prednisolone. In contrast, E2 enhanced μglial staining in Aβ-infused rats. Aβ-immunoreactive (ir) deposits were quantitatively smaller, appeared denser, and were associated with robust μglial responses. Despite the fact that steroid produced a smaller more focal deposit, total extracted Aβ in cortical homogenate was elevated. Together, the in vivo and in vitro data support a role for steroids in plaque compaction. Our data are also consistent with the hypothesis that although E2 is less potent than Gc in impeding Aβ degradation, long term exposure to both steroids could reduce Aβ clearance and clinical utility. These data showing Gc potentiation of Aβ-induced μglial toxins may help explain the lack of epidemiological correlation for AD. The failure of both steroids to accelerate Aβ degradation may explain their lack of efficacy for treatment of AD.     

Isoform-specific effects of ApoE on neurite outgrowth in Olfactory Epithelium culture

Background

The apolipoprotein E4 (apoE4) genotype is a major risk factor for developing late-onset Alzheimer’s disease (AD). Inheritance of apoE4 is also associated with impairments in olfactory function in early stages of AD. In this project we examined the effects of the three common isoforms of human apoE (apoE2, apoE3, and apoE4) on neuronal differentiation and neurite outgrowth in explant cultures of mouse olfactory epithelium (OE).

Results

The OE cultures derived from apoE-deficient/knockout (KO) mice have significantly fewer neurons with shorter neurite outgrowth than cultures from wild-type (WT) mice. Treatment of the apoE KO culture with either purified human apoE2 or with human apoE3 significantly increased neurite outgrowth. In contrast, treatment with apoE4 did not have an effect on neurite outgrowth. The differential effects of human apoE isoforms on neurite outgrowth were abolished by blocking the low-density lipoprotein receptor-related protein (LRP) with lactoferrin and receptor-associated protein (RAP).

Conclusion

ApoE2 and apoE3 stimulate neurite outgrowth in OE cultures by interacting with the lipoprotein receptor, LRP. ApoE4, the isoform associated with AD, failed to promote neurite outgrowth, suggesting a potential mechanism whereby apoE4 may lead to olfactory dysfunction in AD patients.     

Impaired secretion of apolipoprotein E2 from macrophages

Human apoE is a multifunctional and polymorphic protein synthesized and secreted by liver, brain, and tissue macrophages. Here we show that apoE isoforms and mutants expressed through lentiviral transduction display cell-specific differences in secretion efficiency. Whereas apoE3, apoE4, and a natural mutant of apoE4 (apoE-Cys(142)) were efficiently secreted from macrophages, apoE2 and a non-natural apoE mutant (apoE-Cys(112)/Cys(142)) were retained in the perinuclear region and only minimally secreted. The secretory block for apoE2 in macrophages was not affected by the ablation of LDLR (low density lipoprotein receptor), ABCA-1, or SR-BI (scavenger receptor class B type I) but was released in the absence of low density lipoprotein receptor related protein (LRP). In co-immunoprecipitation experiments, an anti-apoE antibody pulled down two times more LRP in apoE2-transduced macrophages than in apoE3-expressing macrophages. Non-reducing SDS-PAGE/Western blot analyses showed that macrophage apoE2 is mostly dimeric and multimeric, whereas apoE3 is predominantly monomeric. ApoE2 retention and multimer formation also occurred in human macrophages derived from the monocyte cell line THP-1. These results were specific for macrophages, as in transduced mouse primary hepatocytes: 1) ApoE2 was secreted as efficiently as apoE3 and apoE4; 2) all isoforms were exclusively in monomeric form; 3) there was no co-immunoprecipitation of apoE and LRP. A microsomal triglyceride transfer protein (MTP) inhibitor nearly deleted apoB100 secretion from hepatocytes without affecting apoE secretion. These data show that macrophages retain apoE2, a highly expressed protein carried by about 8% of the human population. Given the role of locally produced apoE in regulating cholesterol efflux, modulating inflammation, and controlling oxidative stress, this unique property of apoE2 may have important impacts on atherogenesis.     

Low-density lipoprotein receptor represents an apolipoprotein E-independent pathway of Aβ uptake and degradation by astrocytes

Accumulation of the amyloid β (Aβ) peptide within the brain is hypothesized to be one of the main causes underlying the pathogenic events that occur in Alzheimer disease (AD). Consequently, identifying pathways by which Aβ is cleared from the brain is crucial for better understanding of the disease pathogenesis and developing novel therapeutics. Cellular uptake and degradation by glial cells is one means by which Aβ may be cleared from the brain. In the current study, we demonstrate that modulating levels of the low-density lipoprotein receptor (LDLR), a cell surface receptor that regulates the amount of apolipoprotein E (apoE) in the brain, altered both the uptake and degradation of Aβ by astrocytes. Deletion of LDLR caused a decrease in Aβ uptake, whereas increasing LDLR levels significantly enhanced both the uptake and clearance of Aβ. Increasing LDLR levels also enhanced the cellular degradation of Aβ and facilitated the vesicular transport of Aβ to lysosomes. Despite the fact that LDLR regulated the uptake of apoE by astrocytes, we found that the effect of LDLR on Aβ uptake and clearance occurred in the absence of apoE. Finally, we provide evidence that Aβ can directly bind to LDLR, suggesting that an interaction between LDLR and Aβ could be responsible for LDLR-mediated Aβ uptake. Therefore, these results identify LDLR as a receptor that mediates Aβ uptake and clearance by astrocytes, and provide evidence that increasing glial LDLR levels may promote Aβ degradation within the brain.     

The low density lipoprotein receptor-related protein contributes to selective uptake of high density lipoprotein cholesteryl esters by SW872 liposarcoma cells and primary human adipocytes

The concept that selective transfer of high density lipoprotein (HDL)-derived cholesteryl esters (CE) does not require lipoprotein internalization has been challenged recently by evidence that implicates HDL recycling during the selective uptake process. This has prompted us to examine the role of the low density lipoprotein receptor-related protein (LRP) in selective uptake. LRP is an endocytic receptor for lipoprotein lipase (LpL) and apolipoprotein E (apoE) ligands that are able to mediate selective uptake. We report that molecules that interfere with ligand binding to LRP, such as the receptor-associated protein (RAP), suramin, alpha(2)-macroglobulin, or lactoferrin, inhibit HDL-CE selective uptake by human primary adipocytes and SW872 liposarcoma cells by 35-50%. This partial inhibition of selective uptake from total HDL was not due to preferential inhibition of the HDL(2) or HDL(3) subfractions. Selective uptake by the scavenger receptor BI was not inhibited by RAP, excluding its involvement. Furthermore, in SW872 cells in which LRP was reduced to 14% of control levels by stable antisense expression, selective uptake was attenuated by at least 33%, confirming a role for LRP in this process. RAP, alpha(2)-macroglobulin, lactoferrin, and suramin (individually or in paired combinations) also attenuated selective uptake of HDL-CE by primary human adipocytes by about 40%. On the other hand, human skin fibroblasts express LRP abundantly but lack the capacity for selective uptake, demonstrating that other molecules are required. In SW872 cells, exogenous apoE or LpL can facilitate selective uptake but only the apoE-enhanced uptake can be inhibited by RAP, implicating apoE as a likely co-mediator. We discuss the possible mechanisms by which the endocytic receptor, LRP, can mediate selective uptake.     

Effects of coexpression of the LDL receptor and apoE on cholesterol metabolism and atherosclerosis in LDL receptor-deficient mice

The low density lipoprotein receptor (LDLR) plays a major role in regulation of plasma cholesterol levels as a ligand for apolipoprotein B-100 and apolipoprotein E (apoE). Consequently, LDLR-deficient mice fed a Western-type diet develop significant hypercholesterolemia and atherosclerosis. ApoE not only mediates uptake of atherogenic lipoproteins via the LDLR and other cell-surface receptors, but also directly inhibits atherosclerosis. In this study, we examined the hypothesis that coexpression of the LDLR and apoE would have greater effects than either one alone on plasma cholesterol levels and the development of atherosclerosis in LDLR-deficient mice. LDLR-deficient mice fed a Western-type diet for 10 weeks were injected with recombinant adenoviral vectors encoding the genes for human LDLR, human apoE3, both LDLR and apoE3, or lacZ (control). Plasma lipids were analyzed at several time points after vector injection. Six weeks after injection, mice were analyzed for extent of atherosclerosis by two independent methods. As expected, LDLR expression alone induced a significant reduction in plasma cholesterol due to reduced VLDL and LDL cholesterol levels, whereas overexpression of apoE alone did not reduce plasma cholesterol levels. When the LDLR and apoE were coexpressed in this model, the effects on plasma cholesterol levels were no greater than with expression of the LDLR alone. However, coexpression did result in a substantial increase in large apoE-rich HDL particles. In addition, although the combination of cholesterol reduction and apoE expression significantly reduced atherosclerosis, its effects were no greater than with expression of the LDLR or apoE alone. In summary, in this LDLR-deficient mouse model fed a Western-type diet, there was no evidence of an additive effect of expression of the LDLR and apoE on cholesterol reduction or atherosclerosis.     

Low Density Lipoprotein Receptor-related Protein 1 Promotes Anti-apoptotic Signaling in Neurons by Activating Akt Survival Pathway

The low density lipoprotein receptor-related protein 1 (LRP1) is a multi-ligand receptor abundantly expressed in neurons. Previous work has shown that brain LRP1 levels are decreased during aging and in Alzheimer disease. Although mounting evidence has demonstrated a role for LRP1 in the metabolism of apolipoprotein E/lipoprotein and amyloid-β peptide, whether LRP1 also plays a direct role in neuronal survival is not clear. Here, we show that LRP1 expression is critical for the survival of primary neurons under stress conditions including trophic withdrawal, the presence of apoptosis inducers, or amyloid-β-induced neurotoxicity. Using lentiviral short hairpin RNA to knock down endogenous LRP1 expression, we showed that a depletion of LRP1 leads to an activation of caspase-3 and increased neuronal apoptosis, an effect that was rescued by a caspase-3 inhibitor. A correlation between decreased Akt phosphorylation and the activation of caspase-3 was demonstrated in LRP1 knocked down neurons. Notably, LRP1 knockdown decreased insulin receptor levels in primary neurons, suggesting that decreased neuronal survival might be a consequence of an impaired insulin receptor signaling pathway. Correspondingly, both insulin receptor and phospho-Akt levels were decreased in LRP1 forebrain knock-out mice. These results demonstrate that LRP1 mediates anti-apoptotic function in neurons by regulating insulin receptor and the Akt survival pathway and suggest that restoring LRP1 expression in Alzheimer disease brain might be beneficial to inhibiting neurodegeneration.     

Differential functions of members of the low density lipoprotein receptor family suggested by their distinct endocytosis rates

The low density lipoprotein receptor (LDLR) family is composed of a class of cell surface endocytic receptors that recognize extracellular ligands and internalize them for degradation by lysosomes. In addition to LDLR, mammalian members of this family include the LDLR-related protein (LRP), the very low density lipoprotein receptor (VLDLR), the apolipoprotein E receptor-2 (apoER2), and megalin. Herein we have analyzed the endocytic functions of the cytoplasmic tails of these receptors using LRP minireceptors, its chimeric receptor constructs, and full-length VLDLR and apoER2 stably expressed in LRP-null Chinese hamster ovary cells. We find that the initial endocytosis rates mediated by different cytoplasmic tails are significantly different, with half-times of ligand internalization ranging from less than 30 s to more than 8 min. The tail of LRP mediates the highest rate of endocytosis, whereas those of the VLDLR and apoER2 exhibit least endocytosis function. Compared with the tail of LRP, the tails of the LDLR and megalin display significantly lower levels of endocytosis rates. Ligand degradation analyses strongly support differential endocytosis rates initiated by these receptors. Interestingly apoER2, which has recently been shown to mediate intracellular signal transduction, exhibited the lowest level of ligand degradation efficiency. These results thus suggest that the endocytic functions of members of the LDLR family are distinct and that certain receptors in this family may play their main roles in areas other than receptor-mediated endocytosis.