Apolipoprotein E (apoE), a plasma apolipoprotein that plays a central role in lipoprotein metabolism, is localized in the senile plaques, congophilic angiopathy, and neurofibrillary tangles of Alzheimer disease. Late-onset familial and sporadic Alzheimer disease patients have an increased frequency of one of the three common apoE alleles, e4, suggesting apoE4 is associated with increased susceptibility to disease. To follow up on this suggestion, we compared the binding of synthetic amyloid .8 (.3/A4) peptide to purified apoE4 and apoE3, the most common isoform. Both isoforms bound synthetic P/A4 peptide, the primary constituent of the plaque and angiopathy, forming a complex that resisted dissociation by boiling in SDS. Oxygen-mediated complex formation was implicated because binding was increased in oxygenated buffer, reduced in nitrogen-purged buffer, and prevented by reduction with dithiothreitol or 2-mercaptoethanol. Binding of fI/A4 peptide was saturable at 10-4 M peptide and required residues 12-28. Examination of apoE fragments revealed that residues 244-272 are critical for complex formation. Both oxidized apoE4 and apoE3 bound fi/A4 peptide; however, binding to apoE4 was observed in minutes, whereas binding to apoE3 required hours. In addition, apoE4 did not bind (8/A4 peptide at pH < 6.6, whereas apoE3 bound P/A4 peptide from pH 7.6 to 4.6. Together these results indicate differences in the two isoforms in complexing with the f/A4 peptide. Binding of P/A4 peptide by oxidized apoE may determine the sequestration or targeting of either apoE or ./A4 peptide, and isoformspecific differences in apoE binding or oxidation may be involved in the pathogenesis of the intra-and extracellular lesions of Alzheimer disease.

The apoipoprotein E (apoE) The inheritance of apolipoprotein E4 (APOE, gene; apoE, protein) is associated with late-onset familial and sporadic Alzheimer disease (1-3). The allele frequency of APOE4 is greatly increased in patients in late-onset Alzheimer disease families and is also markedly elevated (0.40 ± 0.026 in patients; 0.16 ± 0.027 in aged-matched controls; P < 0.00001) in a series of cases of sporadic Alzheimer disease (2). The increased allele frequency of APOE4 in Alzheimer disease has been confirmed in many studies from several countries. Corder et aL (4) reported that the risk of Alzheimer disease was increased as a function of the inherited dose of APOE4 and that the mean age of onset (i) was lowered with each APOE4 allele (= 68.4 ± 1.2 yr, APOE4/4;Kx= 75.5 + 1.0 yr, APOE3/4; x = 84.3 ± 1.3 yr, APOE3/3) in late-onset families. Inheritance of APOE2 alleles decreased the risk of disease and increased the age of onset (5), so that APOE alleles appear to affect the rate of disease expression (4, 5). ApoE3 is the most common isoform in the general population and contains a single cysteine residue at position 112; apoE4 contains an arginine at this position; additionally apoE2 contains an Arg-158--+ Cys difference (6, 7). Isoform-specific properties ofapoE have been described and include different binding properties with the low-density lipoprotein receptor, with P/A4 peptide, and with lipoprotein particles (8-10).Also, the single cysteine in apoE3 permits disulfide bond formation with itself or other molecules (refs. 11 and 12; for review, see ref. 7). In brain tissue from Alzheimer disease patients, apoE is localized to neuritic plaques, vascular amyloid, and some neurofibrillary tangle-bearing neurons (1, 13, 14). In vitro, P/A4 peptide binds more avidly to apoE4 than to apoE3 (8).A comparison of brain sections from Alzheimer disease patients homozygous for APOE4 and APOE3 has demonstrated an increase in the number of amyloid plaques, the density of (3/A4 peptide immunoreactivity, and the area of brain sections occupied by plaques in APOE4/4 patients (15). Therefore, the well-recognized neuropathological heterogeneity of B/A4 peptide immunoreactivity can be explained by the isoform of apoE inherited by the affected patients. These data suggest that apoE4 decorates 8/A4 peptide-containing plaques better than apoE3 in vivo.Dementia in Alzheimer disease is generally accepted to better correlate with the neurofibrillary pathology than with the extent of P/A4 peptide deposition (16). Neurofibrillary lesions contain paired helical filaments, in which the principal constituent is hyperphosphorylated tau, a microtubuleassociated protein (17-21). Because of the genetic relevance of APOE4 and the presence of immunoreactive apoE in neurons containing neurofibrillary tangles, we examined isoform-specific interactions of apoE with recombinant tau before and after phosphorylation with a brain extract. MATERIALS AND METHODSRecombinant tau-40 (441-aa tau isoform from human brain) (22) was expressed and pur...

Apolipoprotein (apo) E4 is a major risk factor for Alzheimer's disease, and many studies have suggested that apoE has isoformspecific effects on the deposition or clearance of amyloid ␤ (A␤) peptides. We examined the effects of apoE isoforms on the processing of amyloid precursor protein (APP) and on A␤ production in rat neuroblastoma B103 cells stably transfected with human wild-type APP695 (B103-APP). Lipid-poor apoE4 increased A␤ production in B103-APP cells to a greater extent than lipid-poor apoE3 (60% vs. 30%) due to more pronounced stimulation of APP recycling by apoE4 than apoE3. The difference in A␤ production was abolished by preincubating the cells with the receptor-associated protein (25 nM), which blocks the low-density lipoprotein receptorrelated protein (LRP) pathway, or by reducing LRP expression by small interference RNA. The differences were also attenuated by replacing Arg-61 with threonine in apoE4 or pretreating apoE4 with small molecules, both of which abolish apoE4 intramolecular domain interaction. Thus, apoE4 appears to modulate APP processing and A␤ production through both the LRP pathway and domain interaction. These findings provide insights into why apoE4 is associated with increased risk for Alzheimer's disease and may represent a potential target for drug development.Alzheimer's disease ͉ neurodegeneration

Previously, we demonstrated in cultured dorsal root ganglion neurons that, in the presence of ␤-migrating very low density lipoproteins (␤-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 ␤-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 ␤-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 ␤-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 receptorrelated 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.

Human apolipoprotein (apo) E contains an amino-and a carboxyl-terminal domain, which are connected by a hinge region (approximately residues 165 to 215). The interaction of the two domains has been suggested to be responsible for the apoE4-binding preference for very low density lipoproteins (VLDL). In the absence of this interaction in apoE3, the preference is for high density lipoproteins (HDL). To exclude the possibility that the interaction of apoE with other apolipoproteins on the native particles may contribute to the isoform-specific preferences, VLDL-like emulsion particles were incubated with apoE, and the lipid-bound apoE was separated from free apoE on a Superose 6 column. The apoE4 bound more effectively to these particles than did apoE3, indicating that the apoE4 preference for VLDL is due not to interactions with other apolipoproteins but to an intrinsic property of apoE4, likely related to domain interaction. Previously, arginine 61 was shown to be critical for the isoform preferences, suggesting that it interacted with an acidic residue(s) in the carboxyl terminus. Substitution of arginine 61 with lysine did not alter the preference of apoE4 for VLDL, demonstrating that a positive charge rather than a specific requirement for arginine is critical for domain interaction. To identify the acidic residue(s) in the carboxyl terminus interacting with arginine 61, the six acidic residues (244, 245, 255, 266, 270, and 271) in a region known to be important for both lipoprotein association and isoformspecific preferences were substituted individually with alanine in apoE4. Only substitution of glutamic acid 255 altered the preference of apoE4 from VLDL to HDL, indicating that this was the sole residue in the carboxyl terminus that interacts with arginine 61. The participation of the hinge region in domain interaction was examined with internal deletion mutants. Deletion of the residues 186 -202 or 186 -223, representing major portions of the hinge region, had no effect on the apoE4 preference for VLDL. This suggests that the hinge region may act as a spacer that connects the two domains. Further deletion into the carboxyl-terminal domain (to residue 244) results in a loss of apoE4 VLDL binding. These studies establish that interaction of arginine 61 and glutamic acid 255 mediates apoE4 domain interaction. Apolipoprotein (apo)1 E (299 amino acids) plays an important role in lipoprotein metabolism through its interaction with the low density lipoprotein (LDL) receptor (1, 2). The three common isoforms of apoE (apoE2, apoE3, and apoE4) are distinguished from each other by their cysteine/arginine content at two polymorphic sites. Apolipoprotein E3 contains cysteine and arginine at positions 112 and 158, respectively, whereas apoE2 contains cysteine and apoE4 contains arginine at both sites (3).Apolipoprotein E4 is associated with higher plasma cholesterol and LDL concentrations than apoE3 and apoE2 and, as a result, is a risk factor for cardiovascular disease (4 -8). One of the distinct metabolic characterist...

Human apolipoprotein E4 (apoE4) binds preferentially to lower density lipoproteins, including very low density lipoproteins, and is associated with increased risk of atherosclerosis and neurodegenerative disorders, including Alzheimer's disease. This binding preference is the result of the presence of Arg-112, which causes Arg-61 in the amino-terminal domain to interact with Glu-255 in the carboxyl-terminal domain. ApoE2 and apoE3, which have Cys-112, bind preferentially to high density lipoproteins (HDL) and do not display apoE4 domain interaction. Mouse apoE, like apoE4, contains the equivalent of Arg-112 and Glu-255, but lacks the critical Arg-61 equivalent (it contains Thr-61). Thus, mouse apoE does not display apoE4 domain interaction and, as a result, behaves like human apoE3, including preferential binding to HDL. To assess the potential role of apoE4 domain interaction in atherosclerosis and neurodegeneration, we sought to introduce apoE4 domain interaction into mouse apoE. Replacing Thr-61 in mouse apoE with arginine converted the binding preference from HDL to very low density lipoproteins in vitro, suggesting that apoE4 domain interaction could be introduced into mouse apoE in vivo. Using gene targeting in embryonic stem cells, we created mice expressing Arg-61 apoE. Heterozygous Arg-61͞wild-type apoE mice displayed two phenotypes found in human apoE4͞E3 heterozygotes: preferential binding to lower density lipoproteins and reduced abundance of Arg-61 apoE in the plasma, reflecting its more rapid catabolism. These findings demonstrate the successful introduction of apoE4 domain interaction into mouse apoE in vivo. The Arg-61 apoE mouse model will allow the effects of apoE4 domain interaction in lipoprotein metabolism, atherosclerosis, and neurodegeneration to be determined.A polipoprotein E (apoE) is a 34-kDa plasma protein that participates in diverse biological processes, including plasma lipoprotein metabolism (1), intracellular cholesterol utilization (2), cell growth (3), immunoregulation (4-6), and neuronal growth and repair (7). The three common isoforms of human apoE-apoE2, apoE3, and apoE4-differ at two positions (8). ApoE3 has cysteine at position 112 and arginine at position 158, whereas apoE2 has cysteines at both positions and apoE4 has arginines. Metabolically and physiologically, the most prevalent isoform, apoE3, is considered to be normal. ApoE2 has reduced affinity for the low density lipoprotein (LDL) receptor and is associated with type III hyperlipoproteinemia (9). ApoE4 is associated with elevated plasma cholesterol and LDL levels and predisposes to cardiovascular disease and neurodegenerative disorders, including Alzheimer's disease (9-12). The mechanisms of apoE4's effects on lipoprotein metabolism and neurodegeneration remain elusive. Unlike apoE2 and apoE3, apoE4 associates preferentially with very low density lipoproteins (VLDL) (13)(14)(15). This preference contributes to the accelerated catabolism of apoE4 in plasma, which results in lower levels of apoE4 than of apoE3 and ...

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