Zhong-Sheng Ji

Differential Cellular Accumulation/Retention of Apolipoprotein E Mediated by Cell Surface Heparan Sulfate Proteoglycans APOLIPOPROTEINS E3 AND E2 GREATER THAN E4

Isoform-specific effects of apolipoprotein E (apoE) on neurite outgrowth and the cytoskeleton are associated with higher intracellular levels of apoE3 than apoE4 in cultured neurons. The current studies, designed to determine the mechanism for the differential intracellular accumulation or retention of apoE, demonstrate that apoE3- and apoE4-containing β-very low density lipoproteins (β-VLDL) possess similar cell binding and internalization and delivery of cholesterol to the cells. However, as assessed by immunocytochemistry, analysis of extracted cellular proteins, or quantitation of 125I-apoE-enriched β-VLDL, there was a 2–3-fold greater accumulation of apoE3 than apoE4 in Neuro-2a cells, fibroblasts, and hepatocytes (HepG2) after 1–2 h, and this differential was maintained for up to 48 h. ApoE2 also accumulated in Neuro-2a cells to a greater extent than apoE4. The differential effect was mediated by the apoE-enriched β-VLDL and not by free apoE. Neither the low density lipoprotein receptor nor the low density lipoprotein receptor-related protein was responsible for the differential accumulation of apoE3 and apoE4, since cells deficient in either or both of these receptors also displayed the differential accumulation. The effect appears to be mediated primarily by cell surface heparan sulfate proteoglycans (HSPG). The retention of both apoE3 and apoE4 was markedly reduced, and the differential accumulation of apoE3 and apoE4 was eliminated both in mutant Chinese hamster ovary cells that did not express HSPG and in HSPG-expressing cells treated with heparinase. The data suggest that cell surface HSPG directly mediate the uptake of apoE-containing lipoproteins, that the differential accumulation/retention of apoE by cells is mediated via HSPG, and that there is a differential intracellular handling of the specific apoE isoforms.

Lactoferrin binding to heparan sulfate proteoglycans and the LDL receptor-related protein. Further evidence supporting the importance of direct binding of remnant lipoproteins to HSPG.

Bovine lactoferrin inhibits the clearance of remnant lipoproteins from the plasma and competes with the cell-surface binding of apolipoprotein (apo) E-enriched remnants. We established that lactoferrin inhibits remnant binding and uptake by interacting with both heparan sulfate proteoglycans (HSPG) and the low-density lipoprotein receptor-related protein (LRP). The binding of 125I-lactoferrin was inhibited 45% to 60% in HepG2 hepatocytes and wild-type Chinese hamster ovary (CHO) cells treated with heparinase to remove HSPG. In mutant CHO cells (pgsD-677) lacking HSPG, the level of 125I-lactoferrin binding was approximately 50% that seen with wild-type CHO cells; thus, about one half of lactoferrin binding appears to be mediated through cell-surface HSPG. A significant fraction of the residual binding of the lactoferrin appears to be mediated through the LRP. The 39-kd protein known to bind to the LRP and to block ligand interaction inhibited 125I-lactoferrin degradation in wild-type CHO cells by 60% to 65%. The addition of the 39-kd protein plus heparinase treatment reduced the binding by 85% to 90% (this combination blocks direct interaction with both the LRP and HSPG). However, it was also shown that the 39-kd protein bound to HSPG and the LRP. Heparinase treatment of wild-type CHO cells decreased the binding of the 125I-39-kd protein by approximately 40%, and the mutant CHO cells lacking HSPG bound half as much 125I-39-kd protein as wild-type CHO cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein E4 Domain Interaction Mediates Detrimental Effects on Mitochondria and Is a Potential Therapeutic Target for Alzheimer Disease

Apolipoprotein (apo) E4 is the major genetic risk factor for late-onset Alzheimer disease (AD). ApoE4 assumes a pathological conformation through an intramolecular interaction mediated by Arg-61 in the amino-terminal domain and Glu-255 in the carboxyl-terminal domain, referred to as apoE4 domain interaction. Because AD is associated with mitochondrial dysfunction, we examined the effect of apoE4 domain interaction on mitochondrial respiratory function. Steady-state amounts of mitochondrial respiratory complexes were examined in neurons cultured from brain cortices of neuron-specific enolase promoter-driven apoE3 (NSE-apoE3) or apoE4 (NSE-apoE4) transgenic mice. All subunits of mitochondrial respiratory complexes assessed were significantly lower in NSE-apoE4 neurons compared with NSE-apoE3 neurons. However, no significant differences in levels of mitochondrial complexes were detected between astrocytes expressing different apoE isoforms driven by the glial fibrillary acidic protein promoter, leading to our conclusion that the effect of apoE4 is neuron specific. In neuroblastoma Neuro-2A (N2A) cells, apoE4 expression reduced the levels of mitochondrial respiratory complexes I, IV, and V. Complex IV enzymatic activity was also decreased, lowering mitochondrial respiratory capacity. Mutant apoE4 (apoE4-Thr-61) lacking domain interaction did not induce mitochondrial dysfunction in N2A cells, indicating that the effect is specific to apoE4-expressing cells and dependent on domain interaction. Consistent with this finding, treatment of apoE4-expressing N2A cells with a small molecule that disrupts apoE4 domain interaction restored mitochondrial respiratory complex IV levels. These results suggest that pharmacological intervention with small molecules that disrupt apoE4 domain interaction is a potential therapeutic approach for apoE4-carrying AD subjects.

Reactivity of Apolipoprotein E4 and Amyloid β Peptide LYSOSOMAL STABILITY AND NEURODEGENERATION

We previously demonstrated that apolipoprotein E4 (apoE4) potentiates lysosomal leakage and apoptosis induced by amyloid β (Aβ) peptide in cultured Neuro-2a cells and hypothesized that the low pH of lysosomes accentuates the conversion of apoE4 to a molten globule, inducing reactive intermediates capable of destabilizing cellular membranes. Here we report that neutralizing lysosomal pH with bafilomycin or NH4Cl abolished the apoE4 potentiation of Aβ-induced lysosomal leakage and apoptosis in Neuro-2a cells. Consistent with these results, apoE4 at acidic pH bound more avidly to phospholipid vesicles and disrupted them to a greater extent than at pH 7.4. Comparison of “Arctic” mutant Aβ, which forms multimers, and GM6 mutant Aβ, which remains primarily monomeric, showed that aggregation is essential for apoE4 to potentiate Aβ-induced lysosomal leakage and apoptosis. Both apoE4 and Aβ1–42 had to be internalized to exert these effects. Blocking the low density lipoprotein receptor-related protein with small interfering RNA abolished the enhanced effects of apoE4 and Aβ on lysosomes and apoptosis. In cultured Neuro-2a cells, Aβ1–42 increased lysosome formation to a greater extent in apoE3- or apoE4-transfected cells than in Neo-transfected cells, as shown by immunostaining for lysosome-associated membrane protein 1. Similarly, in transgenic mice expressing apoE and amyloid precursor protein, hippocampal neurons displayed increased numbers of lysosomes. Thus, apoE4 and Aβ1–42 may work in concert in neurons to increase lysosome formation while increasing the susceptibility of lysosomal membranes to disruption, release of lysosomal enzymes into the cytosol, and neuronal degeneration.

Increased expression of apolipoprotein E in transgenic rabbits results in reduced levels of very low density lipoproteins and an accumulation of low density lipoproteins in plasma.

Transgenic rabbits expressing human apo E3 were generated to investigate mechanisms by which apo E modulates plasma lipoprotein metabolism. Compared with nontransgenic littermates expressing approximately 3 mg/dl of endogenous rabbit apo E, male transgenic rabbits expressing approximately 13 mg/dl of human apo E had a 35% decrease in total plasma triglycerides that was due to a reduction in VLDL levels and an absence of large VLDL. With its greater content of apo E, transgenic VLDL had an increased binding affinity for the LDL receptor in vitro, and injected chylomicrons were cleared more rapidly by the liver in transgenic rabbits. In contrast to triglyceride changes, transgenic rabbits had a 70% increase in plasma cholesterol levels due to an accumulation of LDL and apo E-rich HDL. Transgenic and control LDL had the same binding affinity for the LDL receptor. Both transgenic and control rabbits had similar LDL receptor levels, but intravenously injected human LDL were cleared more slowly in transgenic rabbits than in controls. Changes in lipoprotein lipolysis did not contribute to the accumulation of LDL or the reduction in VLDL levels. These observations suggest that the increased content of apo E3 on triglyceride-rich remnant lipoproteins in transgenic rabbits confers a greater affinity for cell surface receptors, thereby increasing remnant clearance from plasma. The apo E-rich large remnants appear to compete more effectively than LDL for receptor-mediated binding and clearance, resulting in delayed clearance and the accumulation of LDL in plasma.

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.

Susceptibility to diet-induced atherosclerosis in transgenic mice expressing a dysfunctional human apolipoprotein E(Arg 112,Cys142).

Transgenic mice expressing apolipoprotein (apo) E(Cys 142), a human defective variant of apo E, have elevated levels of plasma cholesterol, triglycerides, and very-low-density lipoproteins (VLDL); beta-VLDL, the biochemical hallmark of the human genetic disease type III hyperlipoproteinemia (HLP), is also present in these mice. This study was designed to determine whether these type III HLP mice have an increased susceptibility to spontaneous or diet-induced atherosclerosis. Three 4-month-old male transgenic mice and three male nontransgenic littermates were assessed for the presence of atherosclerotic lesions in the proximal aorta. No lipid-stained microscopic lesions were visible in the aortas of nontransgenic mice, whereas minimal lesions were observed on the aortic valve stumps of transgenic mice. To magnify the effect of the mutant apo E on the susceptibility of the transgenic animals to atherosclerosis, 8 transgenic and 8 nontransgenic mice were fed a synthetic diet containing 1% cholesterol, 16% fat, and 0.5% cholic acid for 3 months. The diet induced an increase in plasma cholesterol level in both transgenic and nontransgenic mice. However, the increase in plasma cholesterol level in the transgenic mice was all in the VLDL fraction, whereas in nontransgenic mice it was due to increases in both VLDL and high-density lipoprotein (HDL) fractions. Plasma triglyceride levels fell in both groups of mice. After 3 months on the diet, there were compositional changes in the VLDL of both groups, characterized mainly by higher cholesteryl ester content, that resulted in beta-migration on agarose gel electrophoresis. Despite similar VLDL lipid compositions, the extent of atherosclerosis differed markedly in the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein E4-induced mitochondrial dysfunction depends on its structure and can be rescued by a structure corrector

in cholesterol-rich lipid rafts of cell membrane, their activities are affected by the levels of cholesterol in cell membrane. We have shown previously that there was significant cholesterol retention in AD brain as compared to agematched non-demented controls (ND) and cholesterol is capable of stimulating b-and g-secretase activities (Xiong et al. Neurobiol. Dis. 29:422-437, 2008). The objective of this study is to investigate apoE alleles and the levels of cholesterol in Alzheimer’s and ND brains. Methods: Genotyping and cholesterol assays were used to determine apoE alleles and the levels of cholesterol in ND and AD brains, respectively. Results: Nine ND and 19 AD brain samples were used in the study. All NDs carried two apoE3 alleles. One AD patient carried one apoE2 and one apoE3 allele. Eight AD patients carried two apoE3 alleles. Nine AD patients carried one apoE4 and one apoE3 allele. One AD patient carried two apoE4 alleles. Cholesterol assay showed that the level of cholesterol is significantly higher in 19 AD brains (9.7962.24mg/mg protein) than that of 9 ND brains (7.961.11) (t test, p1⁄40.0243). The level of cholesterol in 10 apoE4 carrier AD patients is significantly higher (10.4562.21) than that of 9 NDs (7.961.11) (t test, p1⁄40.0041) but not than that of 9 apoE3 carrier AD patients (9.0661.99) (t test, p1⁄40.0909). The level of cholesterol in 9 apoE3 carrier AD patients is higher but not statistically significant than that of 9 NDs (t test, p1⁄4 0.0728). Conclusions: The results suggest that apoE4 allele is associated with higher brain cholesterol and may contribute to altered cholesterol metabolism in AD brain.

Divergent Metabolism of Apolipoproteins E3 and E4 by Cells

Apolipoprotein (apo-) E is a 299-amino acid, 34-kDa protein that is synthesized and secreted by hepatocytes, macrophages, and astrocytes. It is a component of both plasma and cerebrospinal fluid lipoproteins (Borghini et al, 1995; Mahley, 1988; Pitas, 1997; Pitas et al., 1987). As a component of lipoproteins, apo-E is a ligand for several members of the low density lipoprotein (LDL) receptor gene family, including the LDL receptor itself and the LDL receptor-related protein (LRP) (Krieger and Herz, 1994; Mahley, 1988; Pitas et al., 1979). Apo-E occurs in three common forms that are products of different alleles at the same gene locus (Zannis and Breslow, 1981). The proteins apo-E2, apo-E3, and apo-E4 differ by single amino acid changes at amino acids 112 and 158 (Mahley, 1988). Apo-E2 has cysteine at both positions, apo-E4 has arginine at both positions, and apo-E3 has cysteine at position 112 and arginine at 158 (Mahley, 1988; Weisgraber, 1994). These amino acid substitutions have a profound impact on the metabolic properties of the proteins and their association with disease. Of particular interest is the observation that the apo-E4 allele is overrepresented in subjects with late-onset Alzheimer’s disease (AD) and furthermore that subjects who carry the apo-E4 allele develop AD at an earlier age than those with the apo-E3 allele (Corder et al., 1993; Mayeux et al., 1993; Poirier et al., 1993; Saunders et al., 1993). Apo-E4 is therefore a major risk factor for the development of late-onset AD.