We assessed the isoform-specific effects of apolipoprotein (apo) E on the response of Neuro-2a cells to the amyloid ␤ peptide (A␤1-42). As determined by the intracellular staining pattern and the release of ␤-hexosaminidase into the cytosol, apoE4-transfected cells treated with aggregated A␤1-42 showed a greater tendency toward lysosomal leakage than neo-or apoE3-transfected cells. A␤1-42 caused significantly greater cell death and more than 2-fold greater DNA fragmentation in apoE4-secreting than in apoE3-secreting or control cells. H 2 O 2 or staurosporine enhanced cell death and apoptosis in apoE4-transfected cells but not in apoE3-transfected cells. A caspase-9 inhibitor abolished the potentiation of A␤1-42-induced apoptosis by apoE4. Similar results were obtained with conditioned medium from cells secreting apoE3 or apoE4. Cells preincubated for 4 h with a source of apoE3 or apoE4, followed by removal of apoE from the medium and from the cell surface, still exhibited the isoform-specific response to A␤1-42, indicating that the potentiation of apoptosis required intracellular apoE, presumably in the endosomes or lysosomes. Studies of phospholipid (dimyristoylphosphatidylcholine) bilayer vesicles encapsulating 5-(and-6)-carboxyfluorescein dye showed that apoE4 remodeled and disrupted the phospholipid vesicles to a greater extent than apoE3 or apoE2. In response to A␤1-42, vesicles containing apoE4 were disrupted to a greater extent than those containing apoE3. These findings are consistent with apoE4 forming a reactive molecular intermediate that avidly binds phospholipid and may insert into the lysosomal membrane, destabilizing it and causing lysosomal leakage and apoptosis in response to A␤1-42.Human apolipoprotein (apo) 1 E exists in three major isoforms, apoE2, apoE3, and apoE4, which are encoded by three apoE alleles (⑀2, ⑀3, and ⑀4) (1). The ⑀4 allele is a susceptibility gene for Alzheimer's disease (2-5) and other neurodegenerative disorders (6 -9). The apoE isoforms have differential effects on neurite outgrowth in cultured neurons and on the stability of the intracellular cytoskeleton and microtubular system (10 -14). Recently, we demonstrated that a bioactive form of apoE (E4 to a greater extent than E3) can escape an intracellular membrane compartment, enter the cytosol, and interact with cytoskeletal components to form neurofibrillary tangle-like neuronal inclusions (15). Our studies in transgenic mice have indicated a role for apoE4 in neuronal degeneration and behavioral activity (16,17). In addition, apoE is critical in the deposition of amyloid ␤ peptide (A␤) in transgenic mice overproducing the amyloid precursor protein (18 -20).The neurotoxicity of A␤1-42 appears to be an important factor in the pathogenesis of Alzheimer's disease (for review, see Refs. 21 and 22). A␤1-42 is a proteolytic product of the amyloid precursor protein and is a normal component of plasma and cerebrospinal fluid (23,24). A␤ deposition or amyloid plaque formation is found in Alzheimer's disease cases (25,26) and in t...

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 (NSEapoE4) transgenic mice. All subunits of mitochondrial respiratory complexes assessed were significantly lower in NSEapoE4 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. Apolipoprotein (apo)2 E4 is the major genetic risk factor for late-onset Alzheimer disease (AD) (1-6). The protein sequence of apoE4 (299 amino acids) differs from the other two common isoforms (apoE2, apoE3) at residues 112 or 158; apoE3 has a cysteine at residue 112 and an arginine at residue 158, whereas apoE4 has arginines at both positions and apoE2 has cysteines (3, 4). These differences profoundly affect the tertiary protein structure (3-5). ApoE4 displays an intramolecular interaction between its amino-terminal and carboxylterminal domains (7,8), referred to as apoE4 domain interaction. The domain interaction is mediated by Arg-112 in apoE4, which induces a salt bridge between Arg-61 in the amino-terminal domain and Glu-255 in the carboxyl-terminal domain (7,8). This interaction is decreased in apoE2 and apoE3 due to the presence of Cys-112 (8). Substitution of a threonine for Arg-61 in apoE4 disrupts the domain interaction, converting apoE4 to an apoE3-like conformation (7-9).It is postulated that domain interaction is primarily responsible for the pathogenic effects of apoE4 in AD (3-5). Because AD is known to be associated with mitochondrial dysfunction (10 -...

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 ␤-very low density lipoproteins (␤-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 125 IapoE-enriched ␤-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 ␤-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. Apolipoprotein E (apoE)1 is a component of both plasma and cerebrospinal fluid lipoproteins (1-3). Lipoproteins containing apoE are ligands for members of the low density lipoprotein (LDL) receptor gene family, which includes the LDL receptor, the very low density lipoprotein (VLDL) receptor, the apoE receptor 2, and the LDL receptor-related protein (LRP) (1, 4 -8). Plasma lipoproteins containing apoE participate in lipid transport and in the maintenance of cholesterol homeostasis (1). A similar role has been proposed for cerebrospinal fluid lipoproteins in the central nervous system (2, 9).Apolipoprotein E occurs as three isoforms (apoE2, apoE3, and apoE4) that are products of different alleles at the same gene locus (10). These isoforms differ by single amino acid substitutions at positions 112 and 158 of the 299-amino acid protein (1). Whereas apoE3 has cysteine in position 112 and arginine at position 158, apoE2 has cysteine at both positions, and apoE4 has arginine. These single amino acid changes significantly affect the structure and function of the proteins (1,11,12). Apolipoprotein E2 is associated with the development of type III hyperlipoproteinemia (1). Subjects with apoE4 hav...

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