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.