Stable expression and secretion of apolipoproteins E3 and E4 in mouse neuroblastoma cells produces differential effects on neurite outgrowth

Previously, we demonstrated in cultured dorsal root ganglion neurons that, in the presence of beta-migrating very low density lipoproteins (beta-VLDL), apolipoprotein (apo) E4, but not apoE3, suppresses neurite outgrowth. In the current studies, murine neuroblastoma cells (Neuro-2a) were stably transfected with human apoE3 or apoE4 cDNA, and the effect on neurite outgrowth was examined. The stably transfected cells secreted nanogram quantities of apoE (44-89 ng/mg of cell protein in 48 h). In the absence of lipoproteins, neurite outgrowth was similar in the apoE3- and apoE4-secreting cells. The apoE4-secreting cells, when incubated with beta-VLDL, VLDL, cerebrospinal fluid lipoproteins (d < 1.21 g/ml), or with triglyceride/phospholipid (2.7:1 (w/w)) emulsions, showed a reduction in the number of neurites/cell, a decrease in neurite branching, and an inhibition of neurite extension, whereas in the apoE3-secreting cells in the presence of a lipid source, neurite extension was increased. Uptake of beta-VLDL occurred to a similar extent in both the apoE3- and apoE4-secreting cells. With low density lipoproteins or with dimyristoylphosphatidylcholine emulsions, either alone or complexed with cholesterol, no differential effect on neurite outgrowth was observed. A slight differential effect was observed with apoE-containing high density lipoproteins. The differential effect of apoE3 and apoE4 in the presence of beta-VLDL was blocked by incubation of the cells with heparinase and chlorate, with lactoferrin, or with receptor-associated protein, all of which prevent the uptake of lipoproteins by the low density lipoprotein receptor-related protein (LRP). The data suggest that the secreted and/or cell surface-bound apoE interact with the lipoproteins and facilitate their internalization via the heparan sulfate proteoglycan-LRP pathway. The mechanism by which apoE3 and apoE4 exert differential effects on neurite outgrowth remains speculative. However, the data suggest that apoE4, which has been shown to be associated with late onset familial and sporadic Alzheimer's disease, may inhibit neuronal remodeling and contribute to the progression of the disease

Isoform-specific effects of human apolipoprotein E on brain function revealed in ApoE knockout mice: increased susceptibility of females

Apolipoprotein E (apoE) mediates the redistribution of lipids among cells and is expressed at highest levels in brain and liver. Human apoE exists in three major isoforms encoded by distinct alleles (\u3b52, \u3b53, and \u3b54). Compared with APOE \u3b52 and \u3b53, APOE \u3b54 increases the risk of cognitive impairments, lowers the age of onset of Alzheimer's disease (AD), and decreases the response to AD treatments. Besides age, inheritance of the APOE \u3b54 allele is the most important known risk factor for the development of sporadic AD, the most common form of this illness. Although numerous hypotheses have been advanced, it remains unclear how APOE \u3b54 might affect cognition and increase AD risk. To assess the effects of distinct human apoE isoforms on the brain, we have used the neuron-specific enolase (NSE) promoter to express human apoE3 or apoE4 at similar levels in neurons of transgenic mice lacking endogenous mouse apoE. Compared with NSE-apoE3 mice and wild-type controls, NSE-apoE4 mice showed impairments in learning a water maze task and in vertical exploratory behavior that increased with age and were seen primarily in females. These findings demonstrate that human apoE isoforms have differential effects on brain function in vivo and that the susceptibility to apoE4-induced deficits is critically influenced by age and gender. These results could be pertinent to cognitive impairments observed in human APOE \u3b54 carriers. NSE-apoE mice and similar models may facilitate the preclinical assessment of treatments for apoE-related cognitive deficits

Expression of human apolipoprotein B100 in transgenic mice. Editing of human apolipoprotein B100 mRNA

Apolipoprotein B (apoB) is a large glycoprotein that circulates in plasma as a major constituent of numerous lipoproteins. ApoB exists in two forms: apoB48 and apoB100. ApoB48 is identical in sequence to the N-terminal region of apoB100 and is generated by sequence-specific mRNA editing of the apoB100 transcript. Here, we describe the development of a line of mice expressing a human apoB transgene driven by promoter/enhancer sequences from the transthyretin gene. In these mice, immunodetectable human apoB100 is synthesized by the liver, kidney, and brain. Human apoB100 is found in low concentration (approximately 0.1 mg/dl) in the plasma of the transgenic mice and circulates in the low density lipoprotein fraction. The hepatic human apoB100 transcripts undergo mRNA editing at only slightly lower efficiency than the endogenous mouse apoB100 message. Therefore, there is no absolute species specificity to the apoB100 mRNA editing process

Low-dose expression of a human apolipoprotein E transgene in macrophages restores cholesterol efflux capacity of apolipoprotein E-deficient mouse plasma

Apolipoprotein E- (apoE) deficient (E-/-) mice develop severe hyperlipidemia and diffuse atherosclerosis. Low-dose expression of a human apoE3 transgene in macrophages of apoE-deficient mice (E-/-hTgE+/0), which results in about 5% of wild-type apoE plasma levels, did not correct hyperlipidemia but significantly reduced the extent of atherosclerotic lesions. To investigate the contribution of apoE to reverse cholesterol transport, we compared plasmas of wild-type (E+/+), E-/-, and E-/-hTgE+/0 mice for the appearance of apoE-containing lipoproteins by electrophoresis and their capacity to take up and esterify 3H-labeled cholesterol from radiolabeled fibroblasts or J774 macrophages. Wild-type plasma displayed lipoproteins containing apoE that were the size of high density lipoprotein and that had either electrophoretic alpha or gamma mobilities. Similar particles were also present in E-/-hTgE+/0 plasma. Depending on incubation time, E-/- plasma released 48-74% less 3H-labeled cholesterol from fibroblasts than E+/+ plasma, whereas cholesterol efflux into E-/-hTgE+/0 plasma was only 11-25% lower than into E+/+ plasma. E-/-hTgE+/0 plasma also released 10% more 3H-labeled cholesterol from radiolabeled J774 macrophages than E-/- plasma. E+/+ and E-/-hTgE+/0 plasma each esterified significantly more cell-derived 3H-labeled cholesterol than E-/- plasma. Moreover, E-/- plasma accumulated much smaller proportions of fibroblast-derived 3H-labeled cholesterol in fractions with electrophoretic gamma and alpha mobility than E+/+ and E-/-hTgE+/0 plasma. Thus, low-dose expression of apoE in macrophages nearly restored the cholesterol efflux capacity of apoE-deficient plasma through the formation of apoE-containing particles, which efficiently take up cell-derived cholesterol, and through the increase of cholesterol esterification activity. Thus, macrophage-derived apoE may protect against atherosclerosis by increasing cholesterol efflux from arterial wall cells

Apolipoprotein E: impact of cytoskeletal stability in neurons and the relationship to Alzheimer&apos;s disease

Apolipoproteins E3 and E4 exert differential effects on neuronal growth in vitro. Apolipoprotein E3 supports neurite extension, whereas apolipoprotein E4 does not. These isoform-specific effects may influence the stability of the cytoskeleton and may account for the association of the apolipoprotein E4 isoform with the pathogenesis of Alzheimer's disease

Apolipoprotein A-IMilano. Detection of normal A-I in affected subjects and evidence for a cysteine for arginine substitution in the variant A-I

The A-I(Milano) contains a variant form of apolipoprotein A-I, which unlike normal A-I, has cysteine. We have characterized the A-I(Milano) isoforms in two affected subjects, D.M. and D.G. In these subjects, the two most prominent A-I(Milano) isoforms were displaced from the corresponding normal A-I isoforms by a single charge unit toward the anode, as determined by isoelectric focusing. The amino acid compositions of the purified isoforms, which were separated by either preparative isoelectric focusing or thiopropyl-Sepharose 6B chromatography, indicated that the A-I(Milano) from these subjects contained both a normal A-I (A-I(N)) and a cysteine-containing variant A-I (A-I(Cys)). Furthermore, amino acid analyses suggested that the A-I(Cys) differed from normal A-I by a single cysteine for arginine substitution, which was sufficient to account for the charge difference between the two proteins. Partial sequence analysis revealed that an arginine in normal A-I was replaced by cysteine in the variant A-I at residue 173. Consistent with the amino acid analyses, the cysteine-containing isoforms shifted one charge unit toward the cathode after modification of the cysteine residue with cysteamine. Quantitation of the relative amounts of the A-I(N) and A-I(Cys) in D.M. and D.G. by thiopropyl-Sepharose 6B chromatography revealed that the relative levels were different in each subject. The percentage of the total A-I represented by the A-I(N) in D.M. and D.G. was 16.1 and 25.7%, respectively. The demonstration of variable amounts of normal A-I in A-I(Milano) subjects raises some interesting questions regarding the genetics, regulation, and metabolism of apolipoprotein A-I