Lipoprotein-like Particles Research Articles

(Patho)Physiology of Glycosylphosphatidylinositol-Anchored Proteins II: Intercellular Transfer of Matter (Inheritance?) That Matters

Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) are anchored at the outer leaflet of the plasma membrane (PM) bilayer by covalent linkage to a typical glycolipid and expressed in all eukaryotic organisms so far studied. Lipolytic release from PMs into extracellular compartments and intercellular transfer are regarded as the main (patho)physiological roles exerted by GPI-APs. The intercellular transfer of GPI-APs relies on the complete GPI anchor and is mediated by extracellular vesicles such as microvesicles and exosomes and lipid-free homo- or heteromeric aggregates, and lipoprotein-like particles such as prostasomes and surfactant-like particles, or lipid-containing micelle-like complexes. In mammalian organisms, non-vesicular transfer is controlled by the distance between donor and acceptor cells/tissues; intrinsic conditions such as age, metabolic state, and stress; extrinsic factors such as GPI-binding proteins; hormones such as insulin; and drugs such as anti-diabetic sulfonylureas. It proceeds either “directly” upon close neighborhood or contact of donor and acceptor cells or “indirectly” as a consequence of the induced lipolytic release of GPI-APs from PMs. Those displace from the serum GPI-binding proteins GPI-APs, which have retained the complete anchor, and become assembled in aggregates or micelle-like complexes. Importantly, intercellular transfer of GPI-APs has been shown to induce specific phenotypes such as stimulation of lipid and glycogen synthesis, in cultured human adipocytes, blood cells, and induced pluripotent stem cells. As a consequence, intercellular transfer of GPI-APs should be regarded as non-genetic inheritance of (acquired) features between somatic cells which is based on the biogenesis and transmission of matter such as GPI-APs and “membrane landscapes”, rather than the replication and transmission of information such as DNA. Its operation in mammalian organisms remains to be clarified.

(Patho)Physiology of Glycosylphosphatidylinositol-Anchored Proteins I: Localization at Plasma Membranes and Extracellular Compartments.

Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) are anchored at the outer leaflet of plasma membranes (PMs) of all eukaryotic organisms studied so far by covalent linkage to a highly conserved glycolipid rather than a transmembrane domain. Since their first description, experimental data have been accumulating for the capability of GPI-APs to be released from PMs into the surrounding milieu. It became evident that this release results in distinct arrangements of GPI-APs which are compatible with the aqueous milieu upon loss of their GPI anchor by (proteolytic or lipolytic) cleavage or in the course of shielding of the full-length GPI anchor by incorporation into extracellular vesicles, lipoprotein-like particles and (lyso)phospholipid- and cholesterol-harboring micelle-like complexes or by association with GPI-binding proteins or/and other full-length GPI-APs. In mammalian organisms, the (patho)physiological roles of the released GPI-APs in the extracellular environment, such as blood and tissue cells, depend on the molecular mechanisms of their release as well as the cell types and tissues involved, and are controlled by their removal from circulation. This is accomplished by endocytic uptake by liver cells and/or degradation by GPI-specific phospholipase D in order to bypass potential unwanted effects of the released GPI-APs or their transfer from the releasing donor to acceptor cells (which will be reviewed in a forthcoming manuscript).

Apolipoprotein E4 Effects a Distinct Transcriptomic Profile and Dendritic Arbor Characteristics in Hippocampal Neurons Cultured in vitro.

The APOE gene is diversified by three alleles ε2, ε3, and ε4 encoding corresponding apolipoprotein (apo) E isoforms. Possession of the ε4 allele is signified by increased risks of age-related cognitive decline, Alzheimer’s disease (AD), and the rate of AD dementia progression. ApoE is secreted by astrocytes as high-density lipoprotein-like particles and these are internalized by neurons upon binding to neuron-expressed apoE receptors. ApoE isoforms differentially engage neuronal plasticity through poorly understood mechanisms. We examined here the effects of native apoE lipoproteins produced by immortalized astrocytes homozygous for ε2, ε3, and ε4 alleles on the maturation and the transcriptomic profile of primary hippocampal neurons. Control neurons were grown in the presence of conditioned media from Apoe–/– astrocytes. ApoE2 and apoE3 significantly increase the dendritic arbor branching, the combined neurite length, and the total arbor surface of the hippocampal neurons, while apoE4 fails to produce similar effects and even significantly reduces the combined neurite length compared to the control. ApoE lipoproteins show no systemic effect on dendritic spine density, yet apoE2 and apoE3 increase the mature spines fraction, while apoE4 increases the immature spine fraction. This is associated with opposing effects of apoE2 or apoE3 and apoE4 on the expression of NR1 NMDA receptor subunit and PSD95. There are 1,062 genes differentially expressed across neurons cultured in the presence of apoE lipoproteins compared to the control. KEGG enrichment and gene ontology analyses show apoE2 and apoE3 commonly activate expression of genes involved in neurite branching, and synaptic signaling. In contrast, apoE4 cultured neurons show upregulation of genes related to the glycolipid metabolism, which are involved in dendritic spine turnover, and those which are usually silent in neurons and are related to cell cycle and DNA repair. In conclusion, our work reveals that lipoprotein particles comprised of various apoE isoforms differentially regulate various neuronal arbor characteristics through interaction with neuronal transcriptome. ApoE4 produces a functionally distinct transcriptomic profile, which is associated with attenuated neuronal development. Differential regulation of neuronal transcriptome by apoE isoforms is a newly identified biological mechanism, which has both implication in the development and aging of the CNS.

ApoE and ApoE Nascent-Like HDL Particles at Model Cellular Membranes: Effect of Protein Isoform and Membrane Composition.

Apolipoprotein E (ApoE), an important mediator of lipid transportation in plasma and the nervous system, plays a large role in diseases such as atherosclerosis and Alzheimer's. The major allele variants ApoE3 and ApoE4 differ only by one amino acid. However, this difference has major consequences for the physiological behaviour of each variant. In this paper, we follow (i) the initial interaction of lipid-free ApoE variants with model membranes as a function of lipid saturation, (ii) the formation of reconstituted High-Density Lipoprotein-like particles (rHDL) and their structural characterisation, and (iii) the rHDL ability to exchange lipids with model membranes made of saturated lipids in the presence and absence of cholesterol [1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) with and without 20 mol% cholesterol]. Our neutron reflection results demonstrate that the protein variants interact differently with the model membranes, adopting different protein conformations. Moreover, the ApoE3 structure at the model membrane is sensitive to the level of lipid unsaturation. Small-angle neutron scattering shows that the ApoE containing lipid particles form elliptical disc-like structures, similar in shape but larger than nascent or discoidal HDL based on Apolipoprotein A1 (ApoA1). Neutron reflection shows that ApoE-rHDL do not remove cholesterol but rather exchange saturated lipids, as occurs in the brain. In contrast, ApoA1-containing particles remove and exchange lipids to a greater extent as occurs elsewhere in the body.

Quercetin Lowers Plasma Triglycerides Accompanied by White Adipose Tissue Browning in Diet-Induced Obese Mice.

Obesity and dyslipidemia are major risk factors for the development of cardiovascular diseases (CVD). Quercetin, a natural flavonoid, lowers plasma triglycerides (TG) in human intervention studies, and its intake is associated with lower CVD risk. The aim of this study was to elucidate the mechanism by which quercetin lowers plasma TG levels in diet-induced obesity. C57Bl/6J mice received a high-fat diet (45% of calories derived from fat) with or without quercetin (0.1% w/w) for 12 weeks. Quercetin decreased plasma TG levels from nine weeks onwards (−19%, p < 0.05), without affecting food intake, body composition, or energy expenditure. Mechanistically, quercetin did not reduce intestinal fatty acid (FA) absorption. Rather, quercetin induced a slight reduction in liver Apob expression (−13%, p < 0.05), which suggests decreased very-low density lipoprotein-TG production. Interestingly, quercetin also markedly increased the uptake of [3H]oleate, which was derived from glycerol tri[3H]oleate-labeled lipoprotein-like particles by subcutaneous white adipose tissue (sWAT, +60%, p < 0.05). Furthermore, quercetin also markedly increased mRNA expression of Ucp1 (+229%, p < 0.05) and Elovl3 (+138%, p < 0.05), specifically in sWAT. Accordingly, only quercetin-treated animals showed uncoupling protein-1 protein-positive cells in sWAT, which is fully compatible with increased browning. Taken together, the TG-lowering effect of quercetin may, at least in part, be due to increased TG-derived FA uptake by sWAT as a consequence of browning.

Atorvastatin accelerates clearance of lipoprotein remnants generated by activated brown fat to further reduce hypercholesterolemia and atherosclerosis

Background and aimsActivation of brown adipose tissue (BAT) reduces both hyperlipidemia and atherosclerosis by increasing the uptake of triglyceride-derived fatty acids by BAT, accompanied by formation and clearance of lipoprotein remnants. We tested the hypothesis that the hepatic uptake of lipoprotein remnants generated by BAT activation would be accelerated by concomitant statin treatment, thereby further reducing hypercholesterolemia and atherosclerosis. MethodsAPOE*3-Leiden.CETP mice were fed a Western-type diet and treated without or with the selective β3-adrenergic receptor (AR) agonist CL316,243 that activates BAT, atorvastatin (statin) or both. Resultsβ3-AR agonism increased energy expenditure as a result of an increased fat oxidation by activated BAT, which was not further enhanced by statin addition. Accordingly, statin treatment neither influenced the increased uptake of triglyceride-derived fatty acids from triglyceride-rich lipoprotein-like particles by BAT nor further lowered plasma triglyceride levels induced by β3-AR agonism. Statin treatment increased the hepatic uptake of the formed cholesterol-enriched remnants generated by β3-AR agonism. Consequently, statin treatment further lowered plasma cholesterol levels. Importantly, statin, in addition to β3-AR agonism, also further reduced the atherosclerotic lesion size as compared to β3-AR agonism alone, without altering lesion severity and composition. ConclusionsStatin treatment accelerates the hepatic uptake of remnants generated by BAT activation, thereby increasing the lipid-lowering and anti-atherogenic effects of BAT activation in an additive fashion. We postulate that, in clinical practice, combining statin treatment with BAT activation is a promising new avenue to combat hyperlipidemia and cardiovascular disease.

Depletion of TM6SF2 disturbs membrane lipid composition and dynamics in HuH7 hepatoma cells

A polymorphism of TM6SF2 associates with hepatic lipid accumulation and reduction of triacylglycerol (TAG) secretion, but the function of the encoded protein has remained enigmatic. We studied the effect of stable TM6SF2 knock-down on the lipid content and composition, mitochondrial fatty acid oxidation and organelle structure of HuH7 hepatoma cells. Knock-down of TM6SF2 resulted in intracellular accumulation of TAGs, cholesterol esters, phosphatidylcholine (PC) and phosphatidylethanolamine. In all of these lipid classes, polyunsaturated lipid species were significantly reduced while saturated and monounsaturated species increased their proportions. The PCs encountered relative and absolute arachidonic acid (AA, 20:4n-6) depletion, and AA was also reduced in the total cellular fatty acid pool. Synthesis and turnover of the hepatocellular glycerolipids was enhanced. The TM6SF2 knock-down cells secreted lipoprotein-like particles with a smaller diameter than in the controls, and more lysosome/endosome structures appeared in the knock-down cells. The mitochondrial capacity for palmitate oxidation was significantly reduced. These observations provide novel clues to TM6SF2 function and raise altered mebrane lipid composition and dynamics among the mechanism(s) by which the protein deficiency disturbs hepatic TAG secretion.

Ultrastructural organisation of HCV from the bloodstream of infected patients revealed by electron microscopy after specific immunocapture

ObjectiveHCV particles are associated with very low-density lipoprotein components in chronically infected patients. These hybrid particles, or ‘lipo-viro particles’ (LVPs), are rich in triglycerides, and contain the viral RNA, the...

Mosaic Interdigitated Structure in Nanoparticle-Templated Phospholipid Bilayer Supports Partial Lipidation of Apolipoprotein A-I.

Using gold nanoparticle-templated high-density lipoprotein-like particles as a model, the nanoparticle-templated phospholipid bilayer is studied from the bottom-up. Data support the phospholipids have a mosaic interdigitated structure. The discontinuous lipid milieu supports partial lipidation of apolipoprotein A-I, different from an ordinary phospholipid bilayer, suggesting that synergy between nanoparticle templates and bound phospholipid layers can modulate amphiphilic proteins for desired functions.

Biochemical properties in membrane of rat astrocytes under oxidative stress

Oxidative stress induced by the treatment with 100µM hydrogen peroxide (H2O2) for 10min enhances release of cytosolic proteins along with fibroblast growth factor 1 (FGF-1) from rat astrocytes without inducing apoptosis. FGF-1 promotes the generation of apolipoprotein E-containing high-density lipoprotein-like particles (apoE/HDL) in astrocytes, which contributes to cholesterol homeostasis in the brain. In this work, we studied various effects of oxidative stress on rat astrocyte׳s membrane to understand the mechanism underlying release of cytosolic proteins and FGF-1. The oxidative stress using 100µM H2O2 enhanced lipid release from rat astrocytes in addition to suppression of lipid synthesis. The lipid synthesis, however, was hardly suppressed by H2O2 in the cell lines such as bovine endothelial cells and HepG2 cells from which the release of cytosolic proteins is not increased by H2O2 unlike rat astrocytes. The treatment of rat astrocytes with H2O2 changed the distribution of lipids and proteins in the caveolin-1-rich domain of membrane to the non-raft domain, which was canceled by the pretreatment of cells with low-density lipoproteins (LDL). These findings suggest that oxidative stress induced by H2O2 changes lipid level of the plasma membrane to make the membrane structure fragile in rat astrocytes. The direct treatment with H2O2 of membrane fraction prepared from rat astrocytes did not enhance lipid release from the membrane. The lipid release, however, was enhanced from the isolated membrane fraction, after the cells were treated with H2O2 and incubated in H2O2-free DPBS. Hydrogen peroxide enhanced phosphorylation of protein kinases such as Akt, MEK, and ERK in intact astrocytes without injury and stress. A MEK inhibitor, U0126, suppressed not only the H2O2-induced ERK phosphorylation but also cytosolic protein release from rat astrocytes. These findings suggest that the H2O2-induced release of cytosolic proteins depends on imbalance of lipid level in the membrane through suppressing lipid synthesis and increasing lipid release induced by the intracellular biochemical reaction such as signal transduction generated in intact rat astrocytes treated with H2O2.

Preferential incorporation of shorter and less unsaturated acyl phospholipids into high density lipoprotein-like particles in the ABCA1- and ABCA7-mediated biogenesis with apoA-I

Molecular species of phosphatidylcholine (PC) and sphingomyelin (SPM) were globally analyzed for lipidomics in the nascent high-density lipoprotein (HDL)-like particles generated with human apolipoprotein A-I (apoA-I) form HEK293 cells where either human ATP binding cassette transporter (ABC) A1 or ABCA7 was transfected and overexpressed. SPM/PC ratio was higher in the ABCA1-mediated HDL than ABCA7-mediated HDL likely being related to their cholesterol content, while it was less than the ratio in the cell membrane in either case. Molecular species composition of hydrocarbon chain moiety in each phospholipid in the HDL largely reflected that in the cells the lipoprotein originated in, without remarkable difference between ABCA1 and ABCA7. Further analysis, however, revealed apparent preference for the molecules with shorter hydrocarbon chain length for both PC and SPM in their relative incorporation into HDL by ABCA1 and ABCA7. Likewise, it was in favor for less-unsaturated hydrocarbon chains of PC while this preference was not apparent for SPM. The results are consistent with the view that assembly of HDL particles with extracellular apoA-I is primarily with the cellular phospholipid molecules being regulated in part by their physicochemical nature.

Astrocyte׳s endogenous apoE generates HDL-like lipoproteins using previously synthesized cholesterol through interaction with ABCA1

Apolipoprotein E (apoE) in the brain is predominantly synthesized in and secreted from astrocytes to generate apoE-containing high-density lipoprotein-like particles (apoE/HDL). However, the mechanism underlying generation of apoE/HDL has not been completely understood. The newly synthesized cholesterol, which is synthesized in rat astrocytes within 24h using [3H]-acetate as a cholesterol precursor, was assembled as lipoproteins with densities of 1.12–1.17g/mL (higher density HDL), although apoE was secreted as lipoproteins with lower densities of 1.08–1.12g/mL from the cells. This finding suggests that the newly synthesized cholesterol is released without the association with apoE, which is like that from apoE-deficient mouse (apoE-KO) astrocytes. The cholesterol released from rat astrocytes at 3 days after the onset of its synthesis (previously synthesized cholesterol) was assembled as apoE/HDL with the densities of 1.08–1.12g/mL (lower density HDL). These findings indicate that the endogenous apoE participates in the release of previously synthesized cholesterol but not newly synthesized one. Whereas, exogenously added human apoE induced release of both newly synthesized and previously synthesized cholesterols to generate apoE/HDL with lower density, suggesting that the cellular pool of cholesterol released by endogenous and exogenous apoE is different. The endogenous apoE was distributed in the caveolin-1-rich domain along with ATP-binding cassette transporter A1 (ABCA1) in the membrane fraction and immuno-precipitated using an anti-ABCA1 antibody. However, this is not the case for ABCA1-KO astrocytes. These findings suggest that endogenous apoE generates lower density HDL to produce more lipidated HDL using previously synthesized cholesterol through the interaction with ABCA1 in caveolin-1-rich domain of astrocytes.

In Vivo and In Vitro Effects of an Apolipoprotein E Mimetic Peptide on Amyloid-β Pathology

Apolipoprotein E (ApoE) is the major apolipoprotein present in the high-density lipoprotein-like particles in the central nervous system (CNS). ApoE is involved in various protective functions in CNS including cholesterol transport, anti-inflammatory, and antioxidant effects. An ApoE peptide would be expected to exert protective effects on neuroinflammation. To determine the effects of an ApoE mimetic peptide Ac-hE18A-NH2 on amyloid-β pathology. Using human APP/PS1ΔE9 transgenic mice and in vitro studies, we have evaluated the effect of an ApoE mimetic peptide, Ac-hE18A-NH2, on amyloid plaque deposition and inflammation. Administration of Ac-hE18A-NH2 to APP/PS1ΔE9 mice for 6 weeks (50 μg/mouse, 3 times a week) significantly improved cognition with a concomitant decrease in amyloid plaque deposition and reduced activated microglia and astrocytes, and increased brain ApoE levels. Oligomeric Aβ42 (oAβ42) and oxidized PAPC (ox-PAPC) inhibited secretion of ApoE in U251 cells, a human astrocyte cell line, and this effect was ameliorated in the presence of peptide Ac-hE18A-NH2. The peptide also increased Aβ42 uptake in a cell line of human macrophages. Peptide Ac-hE18A-NH2 attenuates the effects of oxidative stress on ApoE secretion, inhibits amyloid plaque deposition, and thus could be beneficial in the treatment of Alzheimer's disease.

The LXR agonist GW3965 increases apoA-I protein levels in the central nervous system independent of ABCA1

Lipoprotein metabolism in the central nervous system (CNS) is based on high-density lipoprotein-like particles that use apoE as their predominant apolipoprotein rather than apoA-I. Although apoA-I is not expressed in astrocytes and microglia, which produce CNS apoE, apoA-I is reported to be expressed in porcine brain capillary endothelial cells and also crosses the blood–brain barrier (BBB). These mechanisms allow apoA-I to reach concentrations in cerebrospinal fluid (CSF) that are approximately 0.5% of its plasma levels. Recently, apoA-I has been shown to enhance cognitive function and reduce cerebrovascular amyloid deposition in Alzheimer's Disease (AD) mice, raising questions about the regulation and function of apoA-I in the CNS. Peripheral apoA-I metabolism is highly influenced by ABCA1, but less is known about how ABCA1 regulates CNS apoA-I. We report that ABCA1 deficiency leads to greater retention of apoA-I in the CNS than in the periphery. Additionally, treatment of symptomatic AD mice with GW3965, an LXR agonist that stimulates ABCA1 expression, increases apoA-I more dramatically in the CNS compared to the periphery. Furthermore, GW3965-mediated up-regulation of CNS apoA-I is independent of ABCA1. Our results suggest that apoA-I may be regulated by distinct mechanisms on either side of the BBB and that apoA-I may serve to integrate peripheral and CNS lipid metabolism. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945–2010).

Apolipoprotein B100 secretion by cultured ARPE‐19 cells is modulated by alteration of cholesterol levels

Cholesteryl ester rich apolipoprotein B100 (apoB100) lipoproteins accumulate in Bruch's membrane before the development of age-related macular degeneration. It is not known if these lipoproteins come from the circulation or local ocular tissue. Emerging, but incomplete evidence suggests that the retinal pigmented epithelium (RPE) can secrete lipoproteins. The purpose of this investigation was to determine (i) whether human RPE cells synthesize and secrete apoB100, and (ii) whether this secretion is driven by cellular cholesterol, and if so, (iii) whether statins inhibit this response. The established, human derived ARPE-19 cells challenged with 0-0.8 mM oleic acid accumulated cellular cholesterol, but not triglycerides. Oleic acid increased the amount of apoB100 protein recovered from the medium by both western blot analysis and (35) S-radiolabeled immunoprecipitation while negative stain electron microscopy showed lipoprotein-like particles. Of nine statins evaluated, lipophilic statins induced HMG-CoA reductase mRNA expression the most. The lipophilic Cerivastatin (5 μM) reduced cellular cholesterol by 39% and abrogated apoB100 secretion by 3-fold. In contrast, the hydrophilic statin Pravastatin had minimal effect on apoB100 secretion. These data suggest that ARPE-19 cells synthesize and secrete apoB100 lipoproteins, that this secretion is driven by cellular cholesterol, and that statins can inhibit apoB100 secretion by reducing cellular cholesterol.

Regulation of cholesterol efflux by amyloid β secretion

Amyloid beta (Abeta) is a key molecule in the pathogenesis of Alzheimer's disease, but its physiological function remains unclear. Abeta is produced from amyloid precursor protein (APP) by beta- and gamma-secretases, which is enhanced by high levels of cellular cholesterol, so cholesterol is a risk factor for Alzheimer's disease. This linkage led us to hypothesize that Abeta is produced to regulate cellular cholesterol levels in response to high-cholesterol stimulation. Here we show that Abeta production caused a reduction of cellular cholesterol levels in transfected HEK293 cells and neuronal IMR-32 and Neuro2a cells, which was accompanied by an increase in efflux of cholesterol from cells. Fractionation of the culture media by ultracentrifugation and subsequent immunoelectron microscopic observation revealed that Abeta assembled high-density lipoprotein-like particles with cellular cholesterol during its secretion. This assembly was mediated by the ATP-binding cassette transporter A1. APP transgenic and knockout mice exhibited lower and higher levels of cellular cholesterol in their brains, suggesting that Abeta-mediated regulation of cellular cholesterol is physiological. Furthermore, we found that, when injected into mouse cerebral ventricle, reconstituted lipoproteins with Abeta were excreted into the peripheral tissues more efficiently than those without Abeta. This result suggests that Abeta mediates cholesterol transport from the brain to the circulation. We propose, based on these findings, a novel, apolipoprotein-like function for Abeta that is involved in maintenance of cellular and cerebral cholesterol homeostasis.

Knockdown of FABP5 mRNA decreases cellular cholesterol levels and results in decreased apoB100 secretion and triglyceride accumulation in ARPE-19 cells

To maintain normal retinal function, retinal pigment epithelial (RPE) cells engulf photoreceptor outer segments (ROS) enriched in free fatty acids (FFAs). We have previously demonstrated fatty acid-binding protein 5 (FABP5) downregulation in the RPE/choroidal complex in a mouse model of aging and early age-related macular degeneration. FABPs are involved in intracellular transport of FFAs and their targeting to specific metabolic pathways. To elucidate the role of FABP5 in lipid metabolism, the production of the FABP5 protein in a human RPE cell line was inhibited using RNA interference technology. As a result, the levels of cholesterol and cholesterol ester were decreased by about 40%, whereas FFAs and triglycerides were increased by 18 and 67% after siRNA treatment, respectively. Some species of phospholipids were decreased in siRNA-treated cells. Cellular lipid droplets were evident and apoB secretion was decreased by 76% in these cells. Additionally, we discovered that ARPE-19 cells could synthesize and secrete Apolipoprotein B100 (apoB100), which may serve as a backbone structure for the formation of lipoprotein particles in these cells. Our results indicate that FABP5 mRNA knockdown results in the accumulation of cellular triglycerides, decreased cholesterol levels, and reduced secretion of apoB100 protein and lipoprotein-like particles. These observations indicated that FABP5 plays a critical role in lipid metabolism in RPE cells, suggesting that FABP5 downregulation in the RPE/choroid complex in vivo might contribute to aging and early age-related macular degeneration.

Regulation of cholesterol efflux by amyloid β secretion

Amyloid beta (Abeta) is a key molecule in the pathogenesis of Alzheimer's disease, but its physiological function remains unclear. Abeta is produced from amyloid precursor protein (APP) by beta- and gamma-secretases, which is enhanced by high levels of cellular cholesterol, so cholesterol is a risk factor for Alzheimer's disease. This linkage led us to hypothesize that Abeta is produced to regulate cellular cholesterol levels in response to high-cholesterol stimulation. Here we show that Abeta production caused a reduction of cellular cholesterol levels in transfected HEK293 cells and neuronal IMR-32 and Neuro2a cells, which was accompanied by an increase in efflux of cholesterol from cells. Fractionation of the culture media by ultracentrifugation and subsequent immunoelectron microscopic observation revealed that Abeta assembled high-density lipoprotein-like particles with cellular cholesterol during its secretion. This assembly was mediated by the ATP-binding cassette transporter A1. APP transgenic and knockout mice exhibited lower and higher levels of cellular cholesterol in their brains, suggesting that Abeta-mediated regulation of cellular cholesterol is physiological. Furthermore, we found that, when injected into mouse cerebral ventricle, reconstituted lipoproteins with Abeta were excreted into the peripheral tissues more efficiently than those without Abeta. This result suggests that Abeta mediates cholesterol transport from the brain to the circulation. We propose, based on these findings, a novel, apolipoprotein-like function for Abeta that is involved in maintenance of cellular and cerebral cholesterol homeostasis.

Effect of Leucine to Phenylalanine Substitution on the Nonpolar Face of a Class A Amphipathic Helical Peptide on Its Interaction with Lipid: HIGH RESOLUTION SOLUTION NMR STUDIES OF 4F-DIMYRISTOYLPHOSPHATIDYLCHOLINE DISCOIDAL COMPLEX

Model class A amphipathic helical peptides mimic several properties of apolipoprotein A-I (apoA-I), the major protein component of high density lipoproteins. Previously, we reported the NMR structures of Ac-18A-NH(2) (renamed as 2F because of two phenylalanines), the base-line model class A amphipathic helical peptide in the presence of lipid ( Mishra, V. K., Anantharamaiah, G. M., Segrest, J. P., Palgunachari, M. N., Chaddha, M., Simon Sham, S. W., and Krishna, N. R. (2006) J Biol. Chem. 281, 6511-6519 ). Substitution of two Leu residues on the nonpolar face (Leu(3) and Leu(14)) with Phe residues produced the peptide 4F (so named because of four phenylalanines), which has been extensively studied for its anti-inflammatory and antiatherogenic properties. Like 2F, 4F also forms discoidal nascent high density lipoprotein-like particles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Since subtle structural changes in the peptide-lipid complexes have been shown to be responsible for their antiatherogenic properties, we undertook high resolution NMR studies to deduce detailed structure of 4F in 4F.DMPC discs. Like 2F, 4F adopts a well defined amphipathic alpha-helical structure in association with the lipid at a 1:1 peptide/lipid weight ratio. Nuclear Overhauser effect (NOE) spectroscopy revealed a number of intermolecular close contacts between the aromatic residues in the hydrophobic face of the helix and the lipid acyl chain protons. Similar to 2F, the pattern of observed peptide-lipid NOEs is consistent with a parallel orientation of the amphipathic alpha helix, with respect to the plane of the lipid bilayer, on the edge of the disc (the belt model). However, in contrast to 2F in 2F.DMPC, 4F in the 4F.DMPC complex is located closer to the lipid headgroup as evidenced by a number of NOEs between 4F and DMPC headgroup protons. These NOEs are absent in the 2F.DMPC complex. In addition, the conformation of the DMPC sn-3 chain in 4F.DMPC complex is different than in the 2F.DMPC complex as evidenced by the NOE between lipid 2.CH and betaCH(2) protons in 4F.DMPC, but not in 2F.DMPC, complex. Based on the results of this study, we infer that the antiatherogenic properties of 4F may result from its preferential interaction with lipid headgroups.

Lipoprotein Particles of Intraocular Origin in Human Bruch Membrane: An Unusual Lipid Profile

Throughout adulthood, Bruch membrane (BrM) accumulates esterified cholesterol (EC) associated with abundant 60- to 80-nm-diameter lipoprotein-like particles (LLP), putative apolipoprotein B (apoB) lipoproteins secreted by the retinal pigment epithelium (RPE). In the present study, neutral lipid, phospholipids, and retinoid components of human BrM-LLP were assayed. Particles isolated from paired choroids of human donors were subjected to comprehensive lipid profiling (preparative liquid chromatography [LC] gas chromatography [GC]), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), Western blot analysis, and negative stain electron microscopy. Results were compared to plasma lipoproteins isolated from normolipemic volunteers and to conditioned medium from RPE-J cells supplemented with palmitate to induce particle synthesis and secretion. EC was the largest component (32.4+/-7.9 mol%) of BrM-LLP lipids. EC was 11.3-fold more abundant than triglyceride (TG), unlike large apoB lipoproteins in plasma. Of the fatty acids (FA) esterified to cholesterol, linoleate (18:2n6) was the most abundant (41.7+/-4.7 mol%). Retinyl ester (RE) was detectable at picomolar levels in BrM-LLP. Notably scarce in any BrM-LLP lipid class was the photoreceptor-abundant FA docosahexaenoate (DHA, 22:6n3). RPE-J cells synthesized apoB and numerous EC-rich spherical particles. BrM-LLP composition resembles plasma LDL more than it does photoreceptors. An EC-rich core is possible for newly synthesized lipoproteins as well as those processed in plasma. Abundant EC could contribute to a transport barrier in aging and lesion formation in age-related maculopathy (ARM). Analysis of BrM-LLP composition has revealed new aspects of retinal cholesterol and retinoid homeostasis.