Apolipoprotein E fragments present in Alzheimer's disease brains induce neurofibrillary tangle-like intracellular inclusions in neurons.

Human apolipoprotein (apo) E4, a major risk factor for Alzheimer's disease (AD), occurs in amyloid plaques and neurofibrillary tangles (NFTs) in AD brains; however, its role in the pathogenesis of these lesions is unclear. Here we demonstrate that carboxyl-terminal-truncated forms of apoE, which occur in AD brains and cultured neurons, induce intracellular NFT-like inclusions in neurons. These cytosolic inclusions were composed of phosphorylated tau, phosphorylated neurofilaments of high molecular weight, and truncated apoE. Truncated apoE4, especially apoE4(Delta 272--299), induced inclusions in up to 75% of transfected neuronal cells, but not in transfected nonneuronal cells. ApoE4 was more susceptible to truncation than apoE3 and resulted in much greater intracellular inclusion formation. These results suggest that apoE4 preferentially undergoes intracellular processing, creating a bioactive fragment that interacts with cytoskeletal components and induces NFT-like inclusions containing phosphorylated tau and phosphorylated neurofilaments of high molecular weight in neurons.

Overexpression of apolipoprotein E3 in transgenic rabbits causes combined hyperlipidemia by stimulating hepatic VLDL production and impairing VLDL lipolysis.

The differential effects of overexpression of human apolipoprotein (apo) E3 on plasma cholesterol and triglyceride metabolism were investigated in transgenic rabbits expressing low (<10 mg/dL), medium (10 to 20 mg/dL), or high (>20 mg/dL) levels of apoE3. Cholesterol levels increased progressively with increasing levels of apoE3, whereas triglyceride levels were not significantly affected at apoE3 levels up to 20 mg/dL but were markedly increased at levels of apoE3 >20 mg/dL. The medium expressers had marked hypercholesterolemia (up to 3- to 4-fold over nontransgenics), characterized by an increase in low density lipoprotein (LDL) cholesterol, while the low expressers had only slightly increased plasma cholesterol levels. The medium expressers displayed an 18-fold increase in LDL but also had a 2-fold increase in hepatic very low density lipoprotein (VLDL) triglyceride production, an 8-fold increase in VLDL apoB, and a moderate decrease in the ability of the VLDL to be lipolyzed. However, plasma clearance of VLDL was increased, likely because of the increased apoE3 content. The increase in LDL appears to be due to an enhanced competition of VLDL for LDL receptor binding and uptake, resulting in the accumulation of LDL. The combined hyperlipidemia of the apoE3 high expressers (>20 mg/dL) was characterized by a 19-fold increase in LDL cholesterol but also a 4-fold increase in hepatic VLDL triglyceride production associated with a marked elevation of plasma VLDL triglycerides, cholesterol, and apoB100 (4-, 9-, and 25-fold over nontransgenics, respectively). The VLDL from the high expressers was much more enriched in apoE3 and markedly depleted in apoC-II, which contributed to a >60% inhibition of VLDL lipolysis. The combined effects of stimulated VLDL production and impaired VLDL lipolysis accounted for the increases in plasma triglyceride and VLDL concentrations in the apoE3 high expressers. The hyperlipidemic apoE3 rabbits have phenotypes similar to those of familial combined hyperlipidemia, in which VLDL overproduction is a major biochemical feature. Overall, elevated expression of apoE3 appears to determine plasma lipid levels by stimulating hepatic VLDL production, enhancing VLDL clearance, and inhibiting VLDL lipolysis. Thus, the differential expression of apoE may, within a rather narrow range of concentrations, play a critical role in modulating plasma cholesterol and triglyceride levels and may represent an important determinant of specific types of hyperlipoproteinemia.

Chylomicron metabolism in normal, cholesterol-fed, and Watanabe heritable hyperlipidemic rabbits. Saturation of the sequestration step of the remnant clearance pathway.

The plasma clearance of radiolabeled chylomicrons was compared in normal, cholesterol-fed, and Watanabe heritable hyperlipidemic (WHHL) rabbits. Chylomicron clearance was rapid in normal rabbits but was significantly retarded in cholesterol-fed and WHHL rabbits. At 40 min after the injection of chylomicrons, 14-17% of the injected dose remained in the plasma of normal rabbits, whereas approximately 40-50% of the injected dose remained in the plasma of cholesterol-fed and WHHL rabbits. The differences were reflected in the reduced plasma clearance by the liver and bone marrow of the cholesterol-fed and WHHL rabbits. The hyperlipidemic rabbits expressed normal levels of low density lipoprotein (LDL) receptor-related protein/alpha 2-macroglobulin receptor in the liver. In contrast, the hepatic levels of LDL receptors were lower in hyperlipidemic rabbits; as expected, they were significantly lower in WHHL rabbits compared with normal and cholesterol-fed rabbits. Furthermore, it was demonstrated that lipoproteins accumulating in the plasma of the hyperlipidemic rabbits competed for and retarded the clearance of chylomicrons from the plasma. Competition was demonstrated by cross-circulation of normal and cholesterol-fed or normal and WHHL rabbits, in which the rapid influx of plasma containing the accumulated plasma lipoproteins from cholesterol-fed or WHHL rabbits was shown to impair the uptake of chylomicrons by the liver and bone marrow of normal rabbits. These observations were extended by infusing isolated lipoproteins into normal rabbits. The rabbit d < 1.02 g/ml (remnant) fraction and the canine cholesterol-rich high density lipoproteins (HDL) with apolipoprotein E (HDLc) inhibited chylomicron clearance, whereas human LDL and HDL from humans and rabbits did not. We conclude that the low LDL receptor activity in the cholesterol-fed and WHHL rabbits may contribute, at least in part, to the impaired clearance by decreasing remnant uptake and causing the accumulation of chylomicron and/or very low density lipoprotein remnants. The accumulated remnant lipoproteins then compete for and saturate the mechanism responsible for the initial rapid clearance of chylomicrons from the plasma. We speculate that saturation of the initial rapid clearance may occur at the sequestration step, which involves the binding of remnants to heparan sulfate proteoglycans in the space of Disse.

Role of heparan sulfate proteoglycans in the binding and uptake of apolipoprotein E-enriched remnant lipoproteins by cultured cells.

Addition of apolipoprotein (apo) E to rabbit beta-very low density lipoproteins (beta-VLDL) has been shown to result in a marked enhancement of their binding and uptake by various cell types. Apolipoprotein E binds to lipoprotein receptors and proteoglycans. To distinguish between apoE binding to these sites, cells were treated with heparinase. Heparinase treatment of receptor-negative familial hypercholesterolemic (FH) fibroblasts and human hepatoma cells (HepG2) released 30-40% of newly synthesized cell surface 35S-labeled proteoglycans and decreased the binding of beta-VLDL+apoE to FH and normal fibroblasts and HepG2 cells by more than 80%. Furthermore, heparinase treatment significantly decreased the uptake of fluorescently labeled beta-VLDL+apoE by HepG2 cells and decreased cholesteryl ester synthesis in FH fibroblasts by 75%. Likewise, canine chylomicron remnants enriched in apoE demonstrated enhanced binding that was 80% inhibited by heparinase treatment of HepG2 cells. Heparinase treatment did not affect beta-VLDL (without added apoE) or low density lipoprotein (LDL) binding to these cells or the binding activity of beta-VLDL+apoE to the LDL receptor-related protein (LRP) or to the LDL receptor on ligand blots. Chinese hamster ovary (CHO) mutant cells lacking the synthesis of either heparan sulfate (pgsD-677) or all proteoglycans (pgsA-745) did not display any enhanced binding of the beta-VLDL+apoE. By comparison, wild-type CHO cells demonstrated enhanced binding of beta-VLDL+apoE that could be abolished by treatment with heparinase. These mutant cells and wild-type CHO cells possessed a similar amount of LRP, as determined by ligand blot analyses and by alpha 2-macroglobulin binding, and possessed a similar amount of LDL receptor activity, as determined by LDL binding. Therefore, we would interpret these data as showing that heparan sulfate proteoglycan may be involved in the initial binding of the apoE-enriched remnants with the subsequent involvement of the LRP in the uptake of these lipoproteins. It remains to be determined whether the heparan sulfate proteoglycan can function by itself in both the binding and internalization of the apoE-enriched remnants or whether the proteoglycan is part of a complex with LRP that mediates a two-step process, i.e. binding and subsequent internalization by the receptor.

Chylomicron metabolism. Chylomicron uptake by bone marrow in different animal species.

Previously it was shown in rabbits that 20-40% of the injected dose of chylomicrons was cleared from the plasma by perisinusoidal bone marrow macrophages. The present study was undertaken to determine whether the bone marrow of other species also cleared significant amounts of chylomicrons. Canine chylomicrons, labeled in vivo with [14C]cholesterol and [3H] retinol, were injected into marmosets (a small, New World primate), rats, guinea pigs, and dogs. Plasma clearance and tissue uptake of chylomicrons in these species were contrasted with results obtained in rabbits in parallel studies. The chylomicrons were cleared rapidly from the plasma in all animals; the plasma clearance of chylomicrons was faster in rats, guinea pigs, and dogs compared with their clearance from the plasma of rabbits and marmosets. The liver was a major site responsible for the uptake of these lipoproteins in all species. However, as in rabbits, the bone marrow of marmosets accounted for significant levels of chylomicron uptake. The uptake by the marmoset bone marrow ranged from one-fifth to one-half the levels seen in the liver. The marmoset bone marrow also took up chylomicron remnants. Perisinusoidal macrophages protruding through the endothelial cells into the marrow sinuses were responsible for the accumulation of the chylomicrons in the marmoset bone marrow, as determined by electron microscopy. In contrast to marmosets, chylomicron clearance by the bone marrow of rats, guinea pigs, and dogs was much less, and the spleen in rats and guinea pigs took up a large fraction of chylomicrons. The uptake of chylomicrons by the non-human primate (the marmoset), in association with the observation that triglyceride-rich lipoproteins accumulate in bone marrow macrophages in patients with type I, III, or V hyperlipoproteinemia, suggests that in humans the bone marrow may clear chylomicrons from the circulation. It is reasonable to speculate that chylomicrons have a role in the delivery of lipids to the bone marrow as a source of energy and for membrane biosynthesis or in the delivery of fat-soluble vitamins.

Chylomicron-chylomicron remnant clearance by liver and bone marrow in rabbits. Factors that modify tissue-specific uptake.

The metabolism of [14C]cholesterol- and [3H]retinol-labeled chylomicrons obtained from canine thoracic duct or rabbit mesenteric lymph was investigated in normal fasted rabbits. Typically, 70-80% of the chylomicrons injected into the rabbits were cleared from the plasma in 20 min, and their uptake was accounted for principally by the liver and the bone marrow. Surprisingly, the bone marrow was a major site of uptake; the uptake ranged from about half that of the liver to a nearly equal amount. The importance and specificity of chylomicron-chylomicron remnant uptake by the bone marrow were established by demonstrating that (a) bone marrow throughout the body accumulated these lipoproteins, (b) the level of uptake was consistent regardless of how the values were calculated or how the chylomicrons were prepared, (c) the uptake represented specific binding, and (d) radiolabeled intestinal lipoproteins induced in vivo delivered cholesterol and retinol to the marrow. Electron microscopic examination of the rabbit bone marrow established that perisinusoidal macrophages uniquely accounted for the uptake of the chylomicrons. Whereas liver cleared a variety of both triglyceride-rich lipoproteins (chylomicrons, chylomicron remnants, and very low density lipoproteins) and cholesterol-rich lipoproteins (beta-very low density lipoproteins and high density lipoproteins containing apolipoprotein E), bone marrow uptake appeared to be restricted to the triglyceride-rich lipoproteins. More chylomicron remnants (generated in a hepatectomized rabbit) were cleared by the liver than by the bone marrow, and the addition of excess apolipoprotein E to chylomicrons resulted in their preferential uptake by the liver. The role of chylomicron-chylomicron remnant delivery of lipids or lipid-soluble vitamins to rabbit bone marrow is open to speculation, and whether triglyceride-rich lipoprotein uptake occurs to a significant extent in the bone marrow of humans remains to be determined.

Isolation and characterization of the apolipoprotein E receptor from canine and human liver.

Previous results have demonstrated that liver membranes possess two distinct lipoprotein receptors: a low density lipoprotein (LDL) receptor that binds lipoproteins containing either apolipoprotein (apo-) B or apo-E, and an apo-E-specific receptor that binds apo-E-containing lipoproteins, but not the apo-B-containing LDL. This study reports the isolation and purification of apo-B,E(LDL) and apo-E receptors from canine and human liver membranes. The receptors were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and were partially purified by DEAE-cellulose chromatography. The apo-B,E(LDL) receptor was isolated by affinity chromatography on LDL-Sepharose. The apo-E receptor, which did not bind to the LDL-Sepharose column, was then purified by using an HDLc (cholesterol-induced high density lipoprotein)-Sepharose affinity column and an immunoaffinity column. Characterization of the receptors revealed that the hepatic apo-B,E(LDL) receptor is similar to the extrahepatic LDL receptor with an apparent Mr = 130,000 on non-reducing sodium dodecyl sulfate-polyacrylamide gels. The apo-E receptor was found to be distinct from the apo-B,E(LDL) receptor, with an apparent Mr = 56,000. The purified apo-E receptor displayed Ca2+-dependent binding to apo-E-containing lipoproteins and did not bind to LDL or chemically modified apo-E HDLc. Antibodies raised against the apo-B,E(LDL) receptor cross-reacted with the apo-E receptor. However, an antibody prepared against the apo-E receptor did not react with the apo-B,E(LDL) receptor. The apo-E receptor also differed from the apo-B,E(LDL) receptor in amino acid composition, indicating that the apo-E receptor and the apo-B,E(LDL) receptor are two distinct proteins. Immunoblot characterization with anti-apo-E receptor immunoglobulin G indicated that the apo-E receptor is present in the hepatic membranes of man, dogs, rats, and mice and is localized to the rat liver parenchymal cells.