Receptor Binding Domain Of apoE Research Articles

ApoE variant associated with cognitive resilience alters AD associated hyperexcitability in dentate granule neurons

AbstractBackgroundCognitive decline in Alzheimer’s disease (AD) differs based on background genetics even with similar pathology. Using genetic linkage analysis, we identified a coding variant in the receptor binding domain of ApoE that leads to changes in cognitive performance across a genetically diverse population harboring familial (FAD) mutations known as the AD‐BXDs (Neuner et al., 2019). Using CRISPR‐based gene editing, we developed a C57BL/6J (B6) mouse carrying the ApoE allele from the DBA/2J (D2) strain (ApoEE163D) and determined that ApoEE163D mice with 5XFAD transgene (ApoEE163D‐5XFAD) are susceptible to cognitive decline compared to B6‐5XFAD mice (Kaczorowski, et al., 2022).Analysis of hippocampal RNA sequencing profiles from the hippocampus indicates robust transcriptomic changes in dentate gyrus (DG) granule cells associated with cognitive resilience to FAD mutations in the presence of the B6 ApoE genotype. Here we aim to evaluate the functional implications.MethodWe adapted the Patch‐Seq method (Cadwell et al., 2017) to investigate transcriptional and electrophysiological changes within the DG associated with both FAD mutations and ApoE genotype. Whole‐cell patch clamp was used to evaluate intrinsic excitability of granule cells from 14month old B6 mice carrying FAD mutations and ApoEE163D, compared to age matched controls. The cellular contents, including the nucleus, were collected after each recording to characterize the transcriptome, and the cells were filled and stained for downstream analysis of neuronal morphology.ResultWe observed a significant interaction of the 5XFAD and ApoE genotypes on DG granule cell excitability. Specifically, cells from ApoEE163D‐5XFAD required less current stimulus to both elicit a single action potential, and to fire in response to a current ramp (rheobase). Although input resistance was comparable across all 4 groups, the sag ratio of ApoEE163D‐5XFAD was also reduced.ConclusionEnhancement of DG excitability of ApoEE163D‐5XFAD mice may underlie increased seizure susceptibility that has been reported previously in FAD animal models on D2 genetic background and associated with increased risk of seizures in AD patients. Ongoing work will integrate DG excitability with single‐cell RNAseq, morphology, and behavior to dissect mechanisms underlying the modifier effect of ApoE genotype on susceptibility and resilience to cognitive deficits in FAD mutation carriers.

APOE variant in the receptor binding domain confers cognitive resilience to familial Alzheimer's mutations and cell-type specific gene expression changes in the hippocampus.

Cognitive resilience to familial Alzheimer's Disease (FAD) is a phenomenon whereby cognitive functioning is better than predicted based on highly penetrant mutations in APP and PS1. This resilience is likely mediated by unidentified genetic factors, and we hypothesize that these factors provide key targets for treatment and prevention of AD. We previously discovered that the most commonly used background strain in AD model research (C57BL/6J, B6) is resilient to AD-related cognitive decline. Using genetic mapping, we determined that cognitive resilience is associated with a variant in the Apoe receptor binding domain and utilized CRISPR to generate a novel Apoe knockin mouse (Apoe KIB6/E163D ). Contextual fear conditioning (CFC), tau pathology, and single-nuclear RNAseq hippocampi analyses were performed in male and female Apoe KIB6/E163D x 5XFAD progeny that differed by only a single variant in Apoe leading to the E163D base pair change in the protein. We found that, indeed, cognitive resilience to the 5XFAD transgene was conferred in ApoeB6/B6 mice compared to Apoe KIB6/E163D mice tested on CFC. Although minimal differences in hyperphosphorylated tau abundance were observed between 5XFAD-ApoeWT/WT mice compared to 5XFAD-Apoe KIB6/E163D , we uncovered robust differences in the transcriptomes of hippocampal excitatory and inhibitory neurons and microglia using single-nuclear sequencing. Gene enrichment and pathway analyses of differentially expressed genes suggest that the 5XFAD-ApoeB6/B6 genotype promotes cation transport and suppression of amyloid fibril formation in excitatory neurons, promotes genes involved in ribosome assembly and protein translation in inhibitory neurons, and enhances gene expression associated with stress response in microglia. Our results suggest that 5XFAD-ApoeB6/B6 homozygotes exhibit cognitive resilience to causal FAD mutations that involve dynamic regulation of gene networks across multiple cell types, emphasizing the need for well-aligned cell culture models of cognitive resilience to validate targets and screen resilience-based interventions. Future work leveraging in vivo electrophysiology and imaging will establish mechanisms whereby Apoe variants alter neural and synaptic function. Understanding the relationship between protective genetic factors and cognitive resilience are crucial for determining mechanisms of resilience and uncovering novel targets for intervention.

Apolipoprotein Mimetic Peptides as Modulators of Lipoprotein Function.

Apolipoprotein (apo)A-I and apoE are the two protein components that have been extensively investigated for their anti-atherogenic properties. Both apolipoproteins possess amphipathic helical structures, responsible for the solubilization of lipids. While apoA-I possesses class A amphipathic helical structures, apoE possesses a 59 residue long amphipathic helical domain linked to a four helix bundle containing the Arg-rich, 10 residue receptor binding domain. An 18 residue model peptide (18A) was designed to mimic the amphipathic helical domains of apoA-I. This and several analogs were able solubilize phospholipids and, when administered into animal models of atherosclerosis, were able to inhibit lesion formation without any effect on plasma cholesterol levels. These analogs were subsequently termed as apoA-I mimetic peptides. When this peptide (18A) was covalently linked to the Arg-rich receptor binding domain of apoE, the resulting peptide Ac-hE18A-NH2, in which hE refers to the 141-150 Arg-rich region of apoE, dramatically reduced plasma cholesterol in several dyslipidemic animal models, resulting in the reduction of lesion formation. This and several other analogs which were able to dramatically decrease plasma cholesterol, analogous to apoE, were termed as apoE mimetic peptides. These observations developed the field of apolipoprotein mimetic peptides which are involved in interacting with lipoproteins and modulating their function. The present review describes progress made in this field which have culminated in clinical trials in humans for both the apoA-I and apoE mimetic peptides.

The study of the neuroprotective activity of the apolipoprotein E peptide mimetic Cog1410 in transgenic strains of Drosophila melanogaster

The neuroprotective activity of peptide mimetic of apolipoprotein E (apoE) Cog1410, containing the amino acid sequence of the apoE receptor-binding domain, has been investigated in transgenic Drosophila melanogaster strains expressing human APP and beta-secretase genes. Expression of double transgenes caused neuropathological processes typical for Alzheimer’s disease (AD): neurodegeneration, cognitive impairments, and amyloid deposition in the brain. It was shown that Cog1410 reduces neurodegeneration in the brain of transgenic flies and improves cognitive functions (odor recognition). These data suggest that Cog1410 is a potential neuroprotector that can be used in AD treatment.

Human apolipoprotein E peptides inhibit hepatitis C virus entry by blocking virus binding

Hepatitis C virus (HCV) entry is a multiple-step process involving a number of host factors and hence represents a promising target for new antiviral drug development. In search of novel inhibitors of HCV infection, we found that a human apolipoprotein E (apoE) peptide, hEP, containing both a receptor binding fragment and a lipid binding fragment of apoE specifically blocked the entry of cell culture grown HCV (HCVcc) at submicromolar concentrations. hEP caused little cytotoxicity in vitro and remained active even if left 24 hours in cell culture. Interestingly, hEP inhibited neither human immunodeficiency virus (HIV)-HCV pseudotypes (HCVpp) nor HIV and Dengue virus (DENV) infection. Further characterization mapped the anti-HCV activity to a 32-residue region that harbors the receptor binding domain of apoE, but this fragment must contain a cysteine residue at the N-terminus to mediate dimer formation. The anti-HCV activity of the peptide appears to be dependent on both its length and sequence and correlates with its ability to bind lipids. Finally, we demonstrated that the apoE-derived peptides directly blocked the binding of both HCVcc and patient serum-derived virus to hepatoma cells as well as primary human hepatocytes. apoE peptides potently inhibit HCV infection and suggest a direct role of apoE in mediating HCV entry. Our findings also highlight the potential of developing apoE mimetic peptides as novel HCV entry inhibitors by targeting HCV-host interactions.

ApoE mimetic peptide decreases Aβ production in vitro and in vivo

BackgroundApolipoprotein E (apoE) is postulated to affect brain Aβ levels through multiple mechanisms--by altering amyloid precursor protein (APP) processing, Aβ degradation, and Aβ clearance. We previously showed that an apoE-derived peptide containing a double repeat of the receptor-binding region was similarly effective in increasing APP processing in vivo. Here, we further examined whether peptides containing tandem repeats of the apoE receptor-binding region (amino acids 141-149) affected APP trafficking, APP processing, and Aβ production.ResultsWe found that peptides containing a double or triple tandem repeat of the apoE receptor-binding region, LRKLRKRLL, increased cell surface APP and decreased Aβ levels in PS1-overexpressing PS70 cells and in primary neurons. This effect was potentiated by a sequential increase in the number of apoE receptor-binding domain repeats (trimer > dimer > monomer). We previously showed that the apoE dimer increased APP CTF in vivo; to determine whether the dimer also affected secreted APP or Aβ levels, we performed a single hippocampal injection of the apoE dimer in wild-type mice and analyzed its effect on APP processing. We found increased sAPPα and decreased Aβ levels at 24 hrs after treatment, suggesting that the apoE dimer may increase α-secretase cleavage.ConclusionsThese data suggest that small peptides consisting of tandem repeats of the apoE receptor-binding region are sufficient to alter APP trafficking and processing. The potency of these peptides increased with increasing repeats of the receptor binding domain of apoE. In addition, in vivo administration of the apoE peptide (dimer) increased sAPPα and decreased Aβ levels in wild-type mice. Overall, these findings contribute to our understanding of the effects of apoE on APP processing and Aβ production both in vitro and in vivo.

Low-density lipoprotein receptors regulate microglial inflammation through c-Jun N-terminal kinase.

Apolipoprotein E (apoE) has been implicated in modulating the central nervous system (CNS) inflammatory response. However, the molecular mechanisms involved in apoE-dependent immunomodulation are poorly understood. We hypothesize that apoE alters the CNS inflammatory response by signaling via low-density lipoprotein (LDL) receptors in glia. To address this hypothesis, we used a small bioactive peptide formed from the receptor-binding domain of apoE, apoE peptide (EP), to study LDL receptor signaling in microglia. To model glial activation, we treated primary mouse microglia and the microglial cell line BV2 with lipopolysaccharide (LPS) and studied two inflammatory responses: an increase in nitric oxide production (NO) and a decrease in apoE production. We found that treatment of primary microglia and BV2 cells with EP attenuated LPS-induced NO accumulation and apoE reduction in a dose-dependent manner. Using the receptor-associated protein to block ligand binding to members of the LDL receptor family, we found that EP attenuated both of these LPS-induced inflammatory responses via LDL receptors. We studied two intracellular signaling cascades associated with apoE: c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). LPS induced both ERK and JNK activation, whereas EP induced ERK activation, but drastically reduced JNK activation. Inhibition of JNK with SP600125 reduced LPS-induced NO production and apoE reduction in a dose-dependent manner. Treatment of microglia with suboptimal EP in combination with JNK inhibitor enhanced attenuation of LPS-induced NO production. These data suggest that microglial LDL receptors regulate JNK activation, which is necessary for apoE modulation of the inflammatory response.

N-methyl- d-aspartate receptor inhibition by an apolipoprotein E-derived peptide relies on low-density lipoprotein receptor-associated protein

The effects of a synthetic apoE peptide, viz., residues 133–149 (apoE[133–149]), a mimetic that comprises the apoE receptor binding domain, on N-methyl- d-aspartate (NMDA)/glycine-induced ion flow through NMDA receptor (NMDAR) channels, have been investigated. The activity of apoE[133–149] was found to depend on the low-density lipoprotein receptor-related protein (LRP). Competition experiments with receptor-associated protein (RAP) and activated α 2-macroglobulin (α 2M ∗), two proteins that compete for apoE binding to LRP, demonstrate that apoE[133–149] inhibition of NMDAR function is mediated at a locus in LRP that overlaps with the binding sites of RAP and α 2M ∗. A coreceptor of LRP, cell surface heparin sulfate proteoglycan, did not function in this system. Additional electrophysiology experiments demonstrated that the inhibitory potency of apoE[133–149] was threefold greater for NMDAR-transfected wild-type Chinese hamster ovary (CHO) cells compared with NMDAR-transfected CHO cells deficient in LRP. Studies with truncation and replacement variants of the apoE peptide demonstrated that the NMDAR inhibitory properties of these peptides correlate with their binding affinities for LRP. These novel results indicate that apoE functions as an inhibitor of NMDAR ion channels indirectly via LRP, and are suggestive of a participatory role for LRP in NMDAR-based neuropathies.

Two apolipoprotein E mimetic peptides, ApoE(130-149) and ApoE(141-155)2, bind to LRP1.

LRP1 is a cell surface receptor responsible for clearing some 30 known ligands. We have previously shown that each of the three complete LDL receptor-homology domains of the LRP1 extracellular domain (sLRPs) binds apoE-enriched beta-VLDL particles. Here we show that two peptides from the N-terminal receptor binding domain of apoE, which are known to elicit a number of different cellular responses, bind to LRP1. Solution binding assays show that the two peptides, apoE(130-149) and apoE(141-155)(2), interact with each of the sLRPs (2, 3, and 4). Each peptide was found to exhibit the same solution binding characteristics as apoE-enriched beta-VLDL particles. Surface plasmon resonance analyses of the sLRP-apoE peptide interaction show that both peptides bind the sLRPs with K(D) values in the 100 nM range, a value similar to the effective concentration required for observation of the cellular responses. Consistent with results from mutagenesis studies of binding of apoE to LDLR, apoE(130-149,Arg142Glu) bound with a K(D) similar to that of the wild-type sequence, while apoE(130-149,Lys143Glu) showed a 10-fold decrease in K(D). Each of the peptides bound heparin, and heparin competed for sLRP binding.

A two-module region of the low-density lipoprotein receptor sufficient for formation of complexes with apolipoprotein E ligands.

The low-density lipoprotein (LDL) receptor transports two different classes of cholesterol-carrying lipoprotein particles into cells: LDL particles, which contain a single copy of apolipoprotein B-100 (apoB-100), and beta-migrating very low-density lipoprotein (beta-VLDL) particles, which contain multiple copies of apolipoprotein E (apoE). The ligand-binding domain of the receptor lies at its amino-terminal end within seven adjacent LDL-A repeats (LA1-LA7). Although prior work clearly establishes that LA5 is required for high-affinity binding of particles containing apolipoprotein E (apoE), the number of ligand-binding repeats sufficient to bind apoE ligands has not yet been determined. Similarly, uncertainty exists as to whether a single lipid-activated apoE receptor-binding site within a particle is capable of binding to the LDLR with high affinity or whether more than one is required. Here, we establish that the LA4-5 two-repeat pair is sufficient to bind apoE-containing ligands, on the basis of binding studies performed with a series of LDLR-derived "minireceptors" containing up to four repeats. Using single chain multimers of the apoE receptor-binding domain (N-apoE), we also show that more than one receptor-binding site in its lipid-activated conformation is required to bind to the LDLR with high affinity. Thus, in addition to inducing a conformational change in the structure of N-apoE, lipid association enhances the affinity of apoE for the LDLR in part by creating a multivalent ligand.

Quantitation of apoE domains in Alzheimer disease brain suggests a role for apoE in Abeta aggregation.

Apolipoprotein E (apoE) and apoE-derived proteolytic fragments are present in amyloid deposits in Alzheimer disease (AD) and cerebral amyloid angiopathy (CAA). In this study, we examined which apoE fragments are most strongly associated with amyloid deposits and whether apoE receptor binding domains were present. We found that both apoE2- and apoE4-specific residues were present on plaques and blood vessels in AD and CAA. We quantified Abeta plaque burden and apoE plaque burdens in 5 AD brains. ApoE N-terminal-specific and C-terminal-specific antibodies covered 50% and 74% of Abeta plaque burden, respectively (p < 0.003). Double-labeling demonstrated that the plaque cores contained the entire apoE protein, but that outer regions contained only a C-terminal fragment, suggesting a cleavage in the random coil region of apoE. Presence of N- and C-terminal apoE cleavage fragments in brain extracts was confirmed by immunoblotting. The numbers of plaques identified by the apoE N-terminal-specific antibodies and the apoE C-terminal-specific antibody were equal, but were only approximately 60% of the total Abeta plaque number (p < 0.0001). Analysis of the size distribution of Abeta and apoE deposits demonstrated that most of the Abeta-positive, apoE-negative deposits were the smallest deposits (less than 150 microm2). These data suggest that C-terminal residues of apoE bind to Abeta and that apoE may help aid in the progression of small Abeta deposits to larger deposits. Furthermore, the presence of the apoE receptor binding domain in the center of amyloid deposits could affect surrounding cells via chronic interactions with cell surface apoE receptors.

Apolipoprotein E2 (Arg136 → Cys) mutation in the receptor binding domain of apoE is not associated with dominant type III hyperlipoproteinemia

Using apoE phenotyping by immunoblotting and apoE genotyping we identified four heterozygous carriers of a rare apolipoprotein (apo) E2 variant, apoE2 (Arg136 → Cys). ApoE2 (Arg136 → Cys) was not distinct from apoE2 (Arg158 → Cys) by phenotyping, but produced a unique pattern of bands on CfoI restriction typing of a 244 bp apoE gene fragment. Two of the four apoE2 (Arg136 → Cys)/3 heterozygotes had elevated triglycerides, two were normolipidemic. The composition of very low density lipoproteins (VLDL) was normal in each of the four apoE2 (Arg136 → Cys) carriers, regardless of the triglyceride concentrations. None of the apoE2 (Arg136 → Cys) carriers displayed a broad β-band and none revealed β-migrating particles in the VLDL. The two hypertriglyceridemic carriers of apoE2 (Arg136 → Cys) were, therefore, classified as having type IV rather than type III hyperlipoproteinemia. LDL receptor binding activities were studied using recombinant apoE loaded to dimyristoylphosphatidylcholine (DMPC) vesicles and to VLDL and from an apoE-deficient individual. LDL receptor binding of apoE2 (Arg136 → Cys) was 14% of apoE3 and was thus higher than that of apoE2 (Arg158 → Cys). Both apoE2 (Arg136 → Cys) and apoE2 (Arg158 → Cys) displayed substantial heparin binding (61 and 53% of apoE3, respectively). As the dominant apoE variants known so far are characterized by more pronounced reductions of heparin binding, we suggest that apoE2 (Arg136 → Cys) is not associated with dominant expression of type III hyperlipoproteinemia. These findings lend support to the concept that apoE variants predisposing to dominant type III hyperlipoproteinemia differ from recessive mutations by a more severe defect in heparin binding.—März, W., M. M. Hoffmann, H. Scharnagl, E. Fisher, M. Chen, M. Nauck, G. Feussner, and H. Wieland. Apolipoprotein E2 (Arg136 → Cys) mutation in the receptor binding domain of apoE is not associated with dominant type III hyperlipoproteinemia.

Apolipoprotein E (ApoE) peptide regulates tau phosphorylation via two different signaling pathways

Previous studies have shown that treating rat cortical neurons in primary culture with apolipoprotein E (apoE) peptide increased cytoplasmic Ca2+ by 2 mechanisms: 1) an influx of extracellular Ca2+ resulting from the activation of a cell surface Ca2+ channel; and 2) release of Ca2+ from internal Ca2+ stores via a G-protein-coupled pathway (Wang and Gruenstein, 1997). These studies employed a biologically active apoE synthetic peptide (apoEdp) derived from the receptor binding domain of apoE. In the present study we examined whether activation of these 2 signal transduction pathways affects phosphorylation of microtubule-associated protein tau. The levels of tau phosphorylation at thr231, ser235, and ser396 were quantified by ELISA employing monoclonal antibodies PHF-6, SMI33, and PHF-1. ApoEdp treatment resulted in a concentration- and time-dependent dephosphorylation of tau at all 3 phosphorylation sites. The apoEdp-induced dephosphorylation of tau at thr231, and ser235 was dependent on the influx of extracellular Ca2+, while dephosphorylation at ser396 was mediated by a pertusis toxin-sensitive G-protein pathway. The involvement of protein phosphatases in mediating the apoEdp-induced dephosphorylation of tau was examined. Pretreatment with the protein phosphatase 2B inhibitor cyclosporin A blocked the apoEdp-induced dephosphorylation of tau at thr231 and ser235 but not at ser396. Pretreatment with the protein phosophatase 2A/1 inhibitor okadaic acid blocked the apoEdp-induced dephosphorylation of tau at all 3 sites, while pretreatment with the protein phosphates 1 inhibitor tautomycin was without effect. The present study suggests that apoE may affect several Ca2+-associated signal transduction pathways that increase the activity of protein phosphatases 2A and 2B, which in turn dephosphorylate tau.

Identification of a neuronal endocytic pathway activated by an apolipoprotein E (apoE) receptor binding peptide

Apolipoprotein E (apoE) is the only serum apolipoprotein that is also found in the extravascular fluid of the brain, where it is thought to play an important role in lipid transport in the central nervous system. In addition apoE has also been implicated in neural regenerative processes and in the etiology of Alzheimer's disease. Peptides derived from the receptor binding domain of apoE are biologically active and bind to low density lipoprotein (LDL) receptors and LDL receptor related protein. There is, however, no direct evidence that these apoE peptides are able to directly activate the endocytic process, either in the brain or elsewhere. In the present paper, we have used electron microscopy and video imaging fluorescence microscopy to investigate the effects of a peptide derived from the receptor binding domain of human apoE on endocytosis in cultured rat cortical neurons. We have found that this tandem dimer repeat peptide induces neuronal endocytosis via a receptor associated protein sensitive pathway. Although the peptide induces a rise in cytoplasmic calcium, this is not required for the induction of endocytosis. On the other hand, normal processing of the endocytic vesicles does appear to require the elevation of cytoplasmic calcium, since inhibition of the calcium response results in the accumulation of large endocytic vesicles.

Rapid elevation of neuronal cytoplasmic calcium by apolipoprotein E peptide.

Apolipoprotein E (apoE) and certain peptides derived from it have been shown to exert neurotoxic effects in vitro, and apoE has been linked to the etiology of Alzheimer's disease. The mechanisms underlying these toxic and pathological effects are, however, not known. To approach this question, we have studied the effects of apoE peptides on the cytoplasmic calcium ([Ca2+]i) homeostasis of cultured cortical neurons. A tandem dimer repeat peptide (apoEdp) derived from the receptor binding domain of apoE was found to have a potent effect on elevation of [Ca2+]i calcium. The pathway by which apoEdp exerted this effect was shown to involve both the mobilization of intracellular calcium and the influx of extracellular calcium, although the effect on influx was more pronounced. Calcium mobilization occurs via a G-protein-linked phospholipase C (PLC) pathway, whereas calcium influx appears to involve a novel Co2+-sensitive channel. Both the mobilization and the influx of calcium require the binding of the apoE peptide to a membrane receptor because both pathways are blocked by antibody to low-density-lipoprotein receptor-related protein. The data suggest that the neurotoxic effects of apoE may be mediated by a persistent elevation of [Ca2+]i.

Rapid elevation of neuronal cytoplasmic calcium by apolipoprotein E peptide

Apolipoprotein E (apoE) and certain peptides derived from it have been shown to exert neurotoxic effects in vitro, and apoE has been linked to the etiology of Alzheimer's disease. The mechanisms underlying these toxic and pathological effects are, however, not known. To approach this question, we have studied the effects of apoE peptides on the cytoplasmic calcium ([Ca2+]1) homeostasis of cultured cortical neurons. A tandem dimer repeat peptide (apoEdp) derived from the receptor binding domain of apoE was found to have a potent effect on elevation of [Ca2+]1 calcium. The pathway by which apoEdp exerted this effect was shown to involve both the mobilization of intracellular calcium and the influx of extracellular calcium, although the effect on influx was more pronounced. Calcium mobilization occurs via a G-protein-linked phospholipase C (PLC) pathway, whereas calcium influx appears to involve a novel Co2+-sensitive channel. Both the mobilization and the influx of calcium require the binding of the apoE peptide to a membrane receptor because both pathways are blocked by anti-body to low-density-lipoprotein receptor-related protein. The data suggest that the neurotoxic effects of apoE may be mediated by a persistent elevation of [Ca2+]1. J. Cell. Physiol. 173:73–83, 1997. © 1997 Wiley-Liss, Inc.

Apo E variants in patients with type III hyperlipoproteinemia

Type III hyperlipoproteinemia (HLP III) is characterized by the reduced catabolism and accumulation of chylomicron and very low density lipoprotein (VLDL) remnants. Most HLP III patients are homozygous for the apolipoprotein E2 (Cys 112, Cys 158) allele; however, several other mutations at this gene locus have been associated with this HLP. In order to assess the presence of rare apo E variants in our population, we have examined apo E phenotypes by isoelectric focusing (IEF) and genotypes by restriction enzyme analysis of polymerase chain reaction (PCR) amplified DNA in 15 patients with HLP III. Lack of concordance between these two methods was observed in 11 subjects (73.3%). DNA sequencing analysis of the receptor binding domain of the apo E gene in the 11 HLP III patients with discrepancies demonstrated the presence of six carriers of the ϵ3(Arg 136 → Ser) allele and three carriers of the ϵ2(Gly 127 → Asp) allele. Five HLP III patients were apo E2/E2 using IEF, but only 2 of them were ϵ2 homozygous using PCR. Two patients were E3/E3 homozygous with normal DNA sequence in the low density lipoprotein receptor binding domain of apo E. In conclusion, our results show that a number of different apo E genotypes are associated with HLP III in this population. More specifically, mutations at positions 127 and 136 might be frequent in Spain and occur in patients with HLP III.

A newly identified polymorphism in the apolipoprotein E enhancer gene region is associated with Alzheimer's disease and strongly with the epsilon 4 allele

Apolipoprotein E allele 4 (apoE epsilon 4) is a major risk factor for late-onset AD. Inheritance of this allele is associated with an earlier age of onset of dementia in individuals with AD. It is unknown whether other polymorphisms in the apoE gene may influence the effect of apoE epsilon 4 on AD. We screened portions of the promoter enhancer element and of the apoE receptor binding domain for other polymorphisms that could affect risk of AD. In particular, a C/G polymorphism at position +113 of the apoE mRNA in the apoE intron 1 enhancer element (IE1) has been recently identified. We found no other polymorphisms. We studied the relationship of the two alleles of the IE1 polymorphism with AD and found an apparent association between IE1 G and AD (n = 94; p = 0.0515). However, the IE1 G allele is also closely associated with apoE epsilon 4 (p < 0.0001). When the presence of apoE epsilon 4 is covaried, the association between the IE1 G allele and AD is no longer statistically significant (odds ratio = 1.29, 95% confidence interval: 0.44, 3.78). In contrast, epsilon 4 is still highly associated with AD when IE1 G is controlled for (odds ratio = 5.91, 95% confidence interval: 3.29, 10.63). Furthermore, there is no significant association between the age of onset of dementia and the inheritance of the G allele. We believe that the apparent association between IE1 G and AD is a consequence of the association between the epsilon 4 and IE1 G alleles.

Dietary fish oil-induced decrease in low density lipoprotein binding to fibroblasts is mediated by apolipoprotein E.

In a previous study we demonstrated that isocaloric substitution of fish oil (FO) for lard in the diet of cynomolgus monkeys resulted in low density lipoproteins (LDL) that were poorer competitors for binding of a standard 125I-labeled LDL and led to less cholesteryl ester accumulation in skin fibroblasts (Linga, V., et al. 1993. J. Lipid Res. 34: 769-778). The decreased binding and cholesteryl ester accumulation by FO LDL appeared related to the LDL apolipoprotein E (apoE) content. We hypothesized that FO LDL had reduced binding to skin fibroblasts due to a decrease in receptor active apoE. To test this hypothesis and determine the relative contribution of apoE versus apolipoprotein B (apoB) in binding of LDL to skin fibroblasts, LDL from cynomolgus monkeys fed lard or FO-containing diets were isolated, characterized, radioiodinated, and tested for binding in the absence or presence of a 10-fold molar excess of monoclonal antibody to the receptor binding domain of apoE (1D7) or apoB-100 (MB47). FO LDL were smaller, contained less apoE (E/B molar ratio = 0.48 +/- 0.03 vs. 1.85 +/- 0.22; P < 0.001), and had a weaker binding affinity (Kd = 11.3 +/- 1.6 vs. 3.8 +/- 0.80 microgram/ml; P < 0.01) compared to the lard counterparts. Furthermore, the apoE/B molar ratio of LDL appeared inversely related to the Kd for binding to skin fibroblasts. Incubation of LDL with skin fibroblasts in the presence of a 10-fold molar excess of monoclonal antibody directed at the receptor binding domain of apoB-100 (MB47) eliminated 96 +/- 3% of binding of FO LDL, but eliminated only 43 +/- 18% of binding for lard LDL. Incubation with a 10-fold molar excess of monoclonal antibody to the receptor-binding domain of apoE (1D7) eliminated only 23 +/- 6% of FO LDL binding to fibroblasts relative to a no-antibody control, but for lard LDL 44 +/- 11% of binding to fibroblasts was eliminated. Both antibodies together blocked all binding of LDL from both diet groups. In a fluid phase precipitation assay > 75% of the LDL particles from both diet groups was precipitated with saturating amounts of MB47, indicating that the proportion of LDL particles expressing this epitope was the same for both diet groups. The same assay using 1D7 showed approximately 4-fold greater precipitation of LDL in the lard versus FO group.(ABSTRACT TRUNCATED AT 400 WORDS)

Uptake of high density lipoprotein cholesterol ester by HepG2 cells involves apolipoprotein E localized on the cell surface.

High density lipoprotein (HDL) cholesterol ester (CE) is taken up by many cells without simultaneous uptake of HDL apoprotein. The studies described herein demonstrate that the selective uptake of cholesterol ester by HepG2 cells is reduced by antibody directed against the receptor-binding domain of apoE (monoclonal antibody (mAb) 1D7) but not by antibody directed against the NH2-terminal portion of the molecule. The reduction, by 1D7, of HDL cholesteryl ester uptake is not due to apoE acquisition by the labeled HDL preparation or by the transfer of [3H]CE of HDL to apoE-containing lipoproteins and uptake by the apoB/E or apoE receptors. Rather, it appears that mAb 1D7 recognizes apoE localized at the cell surface of HepG2 cells. This conclusion is supported by the fact that: 1) reduction of HDL-CE uptake by HepG2 cells is observed within 15 min after the addition of the antibody-ligand mixture; 2) 1D7 is similarly effective in reducing the selective uptake of HDL-CE when added to the ligand or to the cells; 3) three different anti-apoE mAbs (1D7, 3B7, and 3H1) bind specifically to the surface of the cells. We have also demonstrated that heparin (5 mg/ml) does not reduce the amount of apoE-immunoreactive material bound at the cell surface when added before or after the binding period. 1D7, but not 3B7 or 3H1, binds less in the presence of heparin. The observations are consistent with a localization of apoE on the cell membrane rather than on lipoproteins bound to apoB/E or apoE receptors.