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• W2158965128 abstract "Evidence is presented for the differential effects of two isoforms of apolipoprotein (apo) E, apoE3 and apoE4, on neurite outgrowth and on the cytoskeleton of neuronal cells (Neuro-2a) in culture. In the presence of a lipid source, apoE3 enhances and apoE4 inhibits neurite outgrowth. Immunocytochemical studies demonstrate that there is a higher concentration of apoE3 than apoE4 in both the cell bodies and neurites. Cells treated with apoE4 showed fewer microtubules and a greatly reduced ratio of polymerized to monomeric tubulin than did cells treated with apoE3. The effect of apoE4 on depolymerization of microtubules was shown by biochemical, immunocytochemical, and ultrastructural studies. The depolymerization of microtubules and the inhibition of neurite outgrowth associated with apoE4 suggest a mechanism whereby apoE4, which has been linked to the pathogenesis of Alzheimer's disease, may prevent normal neuronal remodeling from occurring later in life, when this neurodegenerative disorder develops. Evidence is presented for the differential effects of two isoforms of apolipoprotein (apo) E, apoE3 and apoE4, on neurite outgrowth and on the cytoskeleton of neuronal cells (Neuro-2a) in culture. In the presence of a lipid source, apoE3 enhances and apoE4 inhibits neurite outgrowth. Immunocytochemical studies demonstrate that there is a higher concentration of apoE3 than apoE4 in both the cell bodies and neurites. Cells treated with apoE4 showed fewer microtubules and a greatly reduced ratio of polymerized to monomeric tubulin than did cells treated with apoE3. The effect of apoE4 on depolymerization of microtubules was shown by biochemical, immunocytochemical, and ultrastructural studies. The depolymerization of microtubules and the inhibition of neurite outgrowth associated with apoE4 suggest a mechanism whereby apoE4, which has been linked to the pathogenesis of Alzheimer's disease, may prevent normal neuronal remodeling from occurring later in life, when this neurodegenerative disorder develops. INTRODUCTIONApolipoprotein (apo) 1The abbreviations used are: apoapolipoproteinLDLlow density lipoproteinLRPLDL receptor-related proteinDRGdorsal root ganglionNeuro-2amurine neuroblastomaBALB/cmurine fibroblastsDMEMDulbecco's modified Eagle's mediumβ-VLDLβ-migrating very low density lipoproteinsDiI1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyaninePBSphosphate-buffered salineBSAbovine serum albuminPIPES1,4-piperazinediethanesulfonic acid. E is a 34-kDa protein component of lipoproteins that mediates their binding to the low density lipoprotein (LDL) receptor and to the LDL receptor-related protein (LRP)(1Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3353) Google Scholar, 2Herz J. Curr. Opin. Lipidol. 1993; 4: 107-113Crossref Scopus (77) Google Scholar, 3Brown M.S. Herz J. Kowal R.C. Goldstein J.L. Curr. Opin. Lipidol. 1991; 2: 65-72Crossref Scopus (144) Google Scholar, 4Mahley R.W. Innerarity T.L. Rall Jr., S.C. Weisgraber K.H. Taylor J.M. Curr. Opin. Lipidol. 1990; 1: 87-95Crossref Scopus (80) Google Scholar). Apolipoprotein E is a major apolipoprotein in the nervous system, where it is thought to redistribute lipoprotein cholesterol among the neurons and their supporting cells and to maintain cholesterol homeostasis(5Boyles J.K. Pitas R.E. Wilson E. Mahley R.W. Taylor J.M. J. Clin. Invest. 1985; 76: 1501-1513Crossref PubMed Scopus (647) Google Scholar, 6Pitas R.E. Boyles J.K. Lee S.H. Foss D. Mahley R.W. Biochim. Biophys. Acta. 1987; 917: 148-161Crossref PubMed Scopus (567) Google Scholar, 7Pitas R.E. Boyles J.K. Lee S.H. Hui D. Weisgraber K.H. J. Biol. Chem. 1987; 262: 14352-14360Abstract Full Text PDF PubMed Google Scholar). Apart from this function, apoE in the peripheral nervous system functions in the redistribution of lipids during regeneration(8Ignatius M.J. Gebicke-Härter P.J. Skene J.H.P. Schilling J.W. Weisgraber K.H. Mahley R.W. Shooter E.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1125-1129Crossref PubMed Scopus (495) Google Scholar, 9Snipes G.J. McGuire C.B. Norden J.J. Freeman J.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1130-1134Crossref PubMed Scopus (224) Google Scholar, 10Boyles J.K. Zoellner C.D. Anderson L.J. Kosik L.M. Pitas R.E. Weisgraber K.H. Hui D.Y. Mahley R.W. Gebicke-Haerter P.J. Ignatius M.J. Shooter E.M. J. Clin. Invest. 1989; 83: 1015-1031Crossref PubMed Scopus (442) Google Scholar).There are three common isoforms of apoE (apoE2, apoE3, and apoE4) that are the products of three alleles (∊2, ∊3, and ∊4) at a single gene locus on chromosome 19(11Das H.K. McPherson J. Bruns G.A.P. Karathanasis S.K. Breslow J.L. J. Biol. Chem. 1985; 260: 6240-6247Abstract Full Text PDF PubMed Google Scholar). Apolipoprotein E3, the most common isoform, has cysteine and arginine at positions 112 and 158, respectively, whereas apoE2 has cysteine at both of these positions and apoE4 has arginine at both(1Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3353) Google Scholar, 12Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar).Accumulating evidence demonstrates that the apoE4 allele (∊4) is specifically associated with sporadic and familial late-onset Alzheimer's disease and is a major risk factor for the disease (13Corder E.H. Saunders A.M. Strittmatter W.J. Schmechel D.E. Gaskell P.C. Small G.W. Roses A.D. Haines J.L. Pericak-Vance M.A. Science. 1993; 261: 921-923Crossref PubMed Scopus (7163) Google Scholar, 14Mayeux R. Stern Y. Ottman R. Tatemichi T.K. Tang M.-X. Maestre G. Ngai C. Tycko B. Ginsberg H. Ann. Neurol. 1993; 34: 752-754Crossref PubMed Scopus (409) Google Scholar, 15Poirier J. Davignon J. Bouthillier D. Kogan S. Bertrand P. Gauthier S. Lancet. 1993; 342: 697-699Abstract PubMed Scopus (1172) Google Scholar, 16Saunders A.M. Strittmatter W.J. Schmechel D. St. George-Hyslop P.H. Pericak-Vance M.A. Joo S.H. Rosi B.L. Gusella J.F. Crapper-MacLachlan D.R. Alberts M.J. Hulette C. Crain B. Goldgaber D. Roses A.D. Neurology. 1993; 43: 1467-1472Crossref PubMed Google Scholar). In accord with these findings, apoE immunoreactivity is associated with both the amyloid plaques and the intracellular neurofibrillary tangles seen in postmortem examinations of brains from Alzheimer's disease patients(17Namba Y. Tomonaga M. Kawasaki H. Otomo E. Ikeda K. Brain Res. 1991; 541: 163-166Crossref PubMed Scopus (1001) Google Scholar, 18Schmechel D.E. Saunders A.M. Strittmatter W.J. Crain B.J. Hulette C.M. Joo S.H. Pericak-Vance M.A. Goldgaber D. Roses A.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9649-9653Crossref PubMed Scopus (1337) Google Scholar). The mechanism by which apoE4 might contribute to Alzheimer's disease is unknown. However, our recent data demonstrating that apoE4 stunts the outgrowth of neurites from dorsal root ganglion (DRG) neurons suggest that apoE may have a direct effect on neuronal development or remodeling(19Handelmann G.E. Boyles J.K. Weisgraber K.H. Mahley R.W. Pitas R.E. J. Lipid Res. 1992; 33: 1677-1688Abstract Full Text PDF PubMed Google Scholar, 20Nathan B.P. Bellosta S. Sanan D.A. Weisgraber K.H. Mahley R.W. Pitas R.E. Science. 1994; 264: 850-852Crossref PubMed Scopus (728) Google Scholar). In an extension of these previous studies, we have now examined the effects of the apoE isoforms on neurite outgrowth and on the cytoskeleton of Neuro-2a cells, a murine neuroblastoma cell line. Apolipoprotein E4 inhibits neurite outgrowth from these cells, and this isoform-specific effect is associated with depolymerization of microtubules.EXPERIMENTAL PROCEDURESCell LinesMurine neuroblastoma (Neuro-2a) cells and murine fibroblasts (BALB/c) were obtained from American Type Culture Collection (Rockville, MD). Neuro-2a cells were maintained at 37°C in a humidified 5% CO2 incubator in Dulbecco's modified Eagle's medium-nutrient mixture (DMEM/F12; 50%:50%) containing 10% fetal bovine serum, penicillin, and streptomycin. For experiments, Neuro-2a cells were plated in this medium in 6-well plates at 25,000 cells/well. After 3-6 h of incubation, the medium was replaced with N2 medium (DMEM/F12 containing growth supplements) alone(21Bottenstein J.E. Sato G.H. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 514-517Crossref PubMed Scopus (2009) Google Scholar), N2 medium containing rabbit β-migrating very low density lipoproteins (β-VLDL) alone (40 μg cholesterol/ml), or N2 medium containing β-VLDL and either purified human apoE3 (30 μg/ml) or purified human apoE4 (30 μg/ml), and the cells were incubated for an additional 48 h. BALB/c fibroblasts were maintained in DMEM containing 10% fetal bovine serum, penicillin, and streptomycin at 37°C in a humidified 7% CO2 incubator. Experiments with BALB/c cells were performed in DMEM. Rabbit β-VLDL from cholesterol-fed animals were isolated as described(22Kowal R.C. Herz J. Weisgraber K.H. Mahley R.W. Brown M.S. Goldstein J.L. J. Biol. Chem. 1990; 265: 10771-10779Abstract Full Text PDF PubMed Google Scholar), and human apoE was purified from the plasma of apoE3 and apoE4 homozygotes(23Rall Jr., S.C. Weisgraber K.H. Mahley R.W. Methods Enzymol. 1986; 128: 273-287Crossref PubMed Scopus (94) Google Scholar); biological activity was assessed by LDL receptor-binding assay(24Pitas R.E. Innerarity T.L. Mahley R.W. J. Biol. Chem. 1980; 255: 5454-5460Abstract Full Text PDF PubMed Google Scholar).Quantitation of Neurite OutgrowthTo assess neurite outgrowth, Neuro-2a cells were grown in test reagents and then nonspecifically stained with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI) and fixed with 4% paraformaldehyde, as described previously(20Nathan B.P. Bellosta S. Sanan D.A. Weisgraber K.H. Mahley R.W. Pitas R.E. Science. 1994; 264: 850-852Crossref PubMed Scopus (728) Google Scholar). Neurons were imaged in fluorescence mode with a confocal laser scanning system (MRC-600, Bio-Rad), and the images were digitized with an Image-1/AT image analysis system (Universal Images, West Chester, PA)(20Nathan B.P. Bellosta S. Sanan D.A. Weisgraber K.H. Mahley R.W. Pitas R.E. Science. 1994; 264: 850-852Crossref PubMed Scopus (728) Google Scholar). The neuronal images were coded, and the neurite extension (the distance from the cell body to the end of the longest neurite) was measured for each neuron. Approximately 50-60 treatment-responsive cells (cells with at least one neurite longer than the diameter of the cell body) were measured for each treatment condition, and the data were calculated as the percent difference between each treatment group and the matched control (N2 medium alone) for each experiment. The percent differences for the different experiments then were averaged. The value for the N2 medium alone was set at 100%. Data are presented as the mean ± S.E. Statistics were done using Stat View II software.ImmunocytochemistryImmunocytochemistry of apoE was performed on Neuro-2a cells and fibroblasts incubated for 48 h with β-VLDL and either apoE3 or apoE4. Cells were fixed for 3 days at 4°C in PBS containing 3% paraformaldehyde and 0.1% glutaraldehyde and quenched with 150 mM sodium acetate in PBS containing 0.1% milk powder (quench buffer). The cells were further incubated for 15 min at room temperature in quench buffer with or without 0.5% Triton X-100. The cells were then washed with PBS containing 15 mM sodium acetate and 0.1% nonfat dry milk (wash solution) and incubated with a polyclonal antibody to apoE (GHE) at a concentration of 1:1000 in wash solution containing goat serum (1:50 dilution) for 1 h at room temperature followed by extensive washes with wash solution. The secondary antibody (anti-rabbit IgG conjugated to Texas Red, Vector Laboratories, Burlingame, CA) was incubated for 1 h before use at a 1:100 dilution in PBS containing 10% fetal bovine serum. The cells were washed with PBS and coverslipped, and serial optical sections (∼1 μm in thickness) were made using a confocal laser scanning microscope.Immunocytochemistry to detect tubulin was performed on Neuro-2a cells and fibroblasts grown for 48 h in medium alone, with β-VLDL alone, or with β-VLDL and either apoE3 or apoE4. Following incubation with test reagents, the medium was aspirated, and the cells were washed twice with PBS. The cells were fixed for 1 h at room temperature in 10 mM HEPES, pH 7.2, containing 100 mM KCl, 3 mM MgCl2, 300 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, 1 mM EGTA, 0.5% Triton X-100, 2% paraformaldehyde, and 0.1% glutaraldehyde, followed by several quick washes with PBS. The cells were quenched with 0.05 M ammonium chloride in PBS for 5 min at room temperature and blocked for 1 h at room temperature with 3% bovine serum albumin (BSA) in PBS. Immunocytochemistry was performed for 45 min at room temperature, using a monoclonal antibody to β-tubulin (Boehringer Mannheim) at a concentration of 1 μg/ml in PBS containing 1% BSA. Following incubation, the cells were washed five times with PBS containing 0.1% BSA and then incubated for 30 min in the dark with goat anti-mouse IgG (Zymed Laboratories Inc., South San Francisco, CA) conjugated to fluorescein isothiocyanate (10 μl/ml) in PBS containing 1% BSA. Cells were coverslipped, and optical sections of 0.5 μm thickness were made using a Bio-Rad MRC-600 confocal laser scanning microscope; the sections were overlaid to obtain a composite image.For localization of actin, the medium was aspirated, and the cells were washed twice with PBS. Cells were fixed with 3% paraformaldehyde in PBS, washed twice with PBS, and permeabilized for 5 min at room temperature with 0.25% Triton X-100 in PBS containing 1% BSA. The permeabilized cells were washed twice with PBS and incubated for 30 min at room temperature in PBS containing 5 units/ml of rhodamine-labeled phalloidin (Molecular Probes, Eugene, OR). The cells were washed twice with PBS and coverslipped, and optical sections were made as described above.In immunocytochemistry experiments, 15-20% of the cells did not respond to the treatments and were similar to control neurons. These cells may represent cells injured during plating.125I-ApoE Binding AssayThe cells were grown in 12-well plates until they reached half of maximal confluence. Cells were washed with medium and incubated with β-VLDL (40 μg cholesterol/ml) along with 30 μg/ml of either 125I-apoE3 or 125I-apoE4 for 48 h. Following incubation, the medium was removed and the cells were washed four times with PBS containing 0.2% BSA at 4°C. The cells were solubilized with 0.1 N NaOH and assayed for protein, and radioactivity was determined by gamma counting. Apolipoprotein E was iodinated using Bolton-Hunter reagent (Amersham Corp.) according to the manufacturer's instructions. The average specific activity was 50 counts/min/ng apoE.Electron Microscopy of Neuro-2a CellsNeuro-2a cells were incubated with test reagents as described above. Following incubation, the cells were lifted from the plates using 0.05% trypsin and 0.5 mM EDTA and pelleted by centrifugation. Cells were fixed for 1 h with 2.5% glutaraldehyde in 0.1 M cacodylate buffer and post-fixed for 1 h in 2% OsO4. The cells were dehydrated, embedded in Epon 812, sectioned (80 nm) using a Reichert Ultracut E, and stained with uranyl acetate and lead citrate. The cells were photographed using a JEOL CX-100II electron microscope. Consistent results were obtained in three independent experiments performed with fresh preparations of apoE and β-VLDL.Binding of 125I-ApoE to MicrotubulesNeuro-2a cells in a 100-mm plate were grown to confluence as described above, scraped from the plate in PBS, and lysed by sonication in PME buffer, pH 7.2 (80 mM PIPES, 1 mM MgCl2, 1 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin), containing 1 mM GTP. The solution was centrifuged and pelleted cell debris discarded. The supernatant was incubated at 37°C for 1 h to facilitate tubulin polymerization. The resultant pellet of microtubules was resuspended in PME buffer, aliquoted into microfuge tubes, and incubated at 37°C with either 125I-apoE3 (2 μg/ml) or 125I-apoE4 (2 μg/ml). Following incubation, the samples were centrifuged for 1 h at 100,000 × g to separate the free 125I-apoE from that bound to microtubules. The radioactivity associated with the supernatant and pellet was estimated by gamma counting.Immunoblotting of TubulinNeuro-2a cells were incubated with test reagents, and cell extracts were prepared as described above. An aliquot of each extract containing an equal amount of protein was centrifuged through a sucrose cushion (PME + 20% sucrose) at 100,000 × g for 1 h at 37°C to separate the microtubules (polymeric tubulin preparation) from the tubulin (monomeric tubulin preparation). The monomeric tubulin preparation was polymerized for 1 h at 37°C by incubating the sample in PME buffer containing 20% sucrose and 1 mM GTP, followed by centrifugation as described above(25Tiwari S.C. Suprenant K.A. Anal. Biochem. 1993; 215: 96-103Crossref PubMed Scopus (37) Google Scholar). The polymeric tubulin preparation was subjected to a temperature-dependent depolymerization-polymerization cycle to remove cellular debris, followed by centrifugation to obtain the microtubules. Aliquots of the cell extract (total tubulin) and the monomeric and polymeric tubulin preparations were subjected to 12% SDS-polyacrylamide gel electrophoresis under reducing conditions and immunoblotted using a monoclonal antibody to α-tubulin (ICN, Irvine, CA) at a dilution of 1:100, as described by the manufacturer. The tubulin bands in the immunoblots were quantitated by densitometry (Ambis Systems, San Diego, CA). The value obtained with N2 medium alone was set at 100%, and the data were calculated as the percent difference between each treatment group and the matched control (N2 medium alone) for each experiment. The percent differences for the different experiments then were averaged. Data are presented as the mean ± S.E. Statistics were done using Stat View II software.Other Assays and MethodsProtein assay was performed as described(26Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). The amount of cholesterol in cells incubated for 48 h with β-VLDL (40 μg cholesterol/ml) and human apoE3 or apoE4 (30 μg/ml) was assayed using a commercially available kit (Monotest, Boehringer Mannheim). The [3H]thymidine incorporation assay was performed with Neuro-2a cells incubated with β-VLDL and apoE3 or apoE4 using a previously published procedure(27Browning P.J. Roberts D.D. Zabrenetzky V. Bryant J. Kaplan M. Washington R.H. Panet A. Gallo R.C. Vogel T. J. Exp. Med. 1994; 180: 1949-1954Crossref PubMed Scopus (30) Google Scholar). The lactate dehydrogenase assay was performed as described(28Koh J.Y. Choi D.W. J. Neurosci. Methods. 1987; 20: 83-90Crossref PubMed Scopus (1234) Google Scholar). The DiI-labeled β-VLDL uptake was performed as described(19Handelmann G.E. Boyles J.K. Weisgraber K.H. Mahley R.W. Pitas R.E. J. Lipid Res. 1992; 33: 1677-1688Abstract Full Text PDF PubMed Google Scholar).RESULTSDifferential Effects of ApoE3 and ApoE4 on Neurite OutgrowthOur previous studies examined the effects of apoE and lipoproteins on the outgrowth of neurites from primary rabbit DRG neurons in vitro(19Handelmann G.E. Boyles J.K. Weisgraber K.H. Mahley R.W. Pitas R.E. J. Lipid Res. 1992; 33: 1677-1688Abstract Full Text PDF PubMed Google Scholar, 20Nathan B.P. Bellosta S. Sanan D.A. Weisgraber K.H. Mahley R.W. Pitas R.E. Science. 1994; 264: 850-852Crossref PubMed Scopus (728) Google Scholar). The addition of purified human apoE3, together with rabbit β-VLDL (cholesterol-rich lipoproteins), increased neurite extension from these peripheral nervous system neurons, whereas apoE4 when added to the cells together with β-VLDL inhibited neurite outgrowth.In this study, we found a similar differential effect of apoE3 and apoE4 on the outgrowth of neurites from Neuro-2a cells. In the presence of β-VLDL, apoE3 and apoE4 had dramatic isoform-specific effects on neurite outgrowth from Neuro-2a cells, as assessed by phase contrast microscopy (Fig. 1). Incubation of the cells with β-VLDL (Fig. 1B) stimulated neurite outgrowth slightly as compared with cells grown in N2 medium alone (Fig. 1A); however, a more dramatic effect was seen with the addition of apoE. Cells incubated with apoE3 and β-VLDL (Fig. 1C) had more neurite extension than cells incubated with β-VLDL, whereas cells incubated with apoE4 and β-VLDL had less neurite extension (Fig. 1D). These observations were confirmed when neurite outgrowth was quantitated using an image analysis system (Fig. 2). Incubation of the cells with β-VLDL enhanced neurite extension, as compared with cells maintained in N2 medium alone (Fig. 2). Addition of human apoE3 with β-VLDL further increased the extension (p < 0.005), whereas human apoE4 along with β-VLDL significantly reduced neurite extension as compared with the extension observed from cells incubated with β-VLDL (p < 0.001).Figure 2:Quantitation of the effect of apoE on neurite extension from Neuro-2a cells. Neuro-2a cells were incubated with test reagents as described in the legend to Fig. 1. Neurite extension then was measured for 50-60 neurons from each group as described under “Experimental Procedures.” Data were calculated as the percent difference between each treatment group and the matched control (N2 medium alone) for each experiment. The percent differences for the various experiments then were averaged. The value for the N2 medium alone was set at 100% (dashed line). Data are presented as the mean ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Three different studies were performed to rule out a general toxic effect of apoE4 on neurons. We assayed lactate dehydrogenase activity, a commonly used indicator of cell death, measured thymidine incorporation into DNA as an indication of cell replication, and examined the ability of the cells to develop neurites following incubation with apoE4 and β-VLDL. No significant differences in lactate dehydrogenase activity (apoE3 +β-VLDL, 212 ± 26.2 units/ml; apoE4 +β-VLDL, 205 ± 22 units/ml) or [3H]thymidine incorporation into DNA (apoE3 +β-VLDL, 17.37 ± 1.66 counts/min × 106; apoE4 +β-VLDL, 18.13 ± 1.42 counts/min × 106) were seen with cells incubated with β-VLDL and either apoE3 or apoE4. The inhibitory effect of apoE4 was reversible, since removal of apoE4 and β-VLDL from the cells in culture and addition of medium alone or medium containing β-VLDL resulted in normal outgrowth (data not shown). These findings taken together demonstrate that the inhibitory effect of apoE4 on neurite outgrowth is not due to a cytotoxic effect of apoE4 on the cells.To determine if lipoproteins were required for the differential effects of apoE3 and apoE4, the cells were incubated with free apoE for 48 h and the effects on neurite outgrowth examined. In the absence of lipoprotein, neither apoE3 nor apoE4 had an effect on neurite extension (data not shown). This result suggests that receptor-mediated endocytosis of apoE is necessary for the differential effects on neurite outgrowth, as previous studies have shown that apoE is a ligand for the LDL receptor and the LRP only when it is present on lipoproteins(1Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3353) Google Scholar, 2Herz J. Curr. Opin. Lipidol. 1993; 4: 107-113Crossref Scopus (77) Google Scholar, 3Brown M.S. Herz J. Kowal R.C. Goldstein J.L. Curr. Opin. Lipidol. 1991; 2: 65-72Crossref Scopus (144) Google Scholar, 29Mahley R.W. Hussain M.M. Curr. Opin. Lipidol. 1991; 2: 170-176Crossref Scopus (104) Google Scholar).Metabolism and Localization of ApoE3 and ApoE4 in Neuro-2a CellsIn several nonneuronal cell lines it has been demonstrated that apoE-containing lipoproteins follow a classic receptor-mediated endocytotic pathway by which the ligands are delivered to lysosomes where they are degraded(1Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3353) Google Scholar, 2Herz J. Curr. Opin. Lipidol. 1993; 4: 107-113Crossref Scopus (77) Google Scholar, 3Brown M.S. Herz J. Kowal R.C. Goldstein J.L. Curr. Opin. Lipidol. 1991; 2: 65-72Crossref Scopus (144) Google Scholar, 12Weisgraber K.H. Adv. Protein Chem. 1994; 45: 249-302Crossref PubMed Google Scholar). Both apoE3- and apoE4-containing lipoproteins exhibit similar binding activity in nonneuronal cells(1Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3353) Google Scholar, 22Kowal R.C. Herz J. Weisgraber K.H. Mahley R.W. Brown M.S. Goldstein J.L. J. Biol. Chem. 1990; 265: 10771-10779Abstract Full Text PDF PubMed Google Scholar, 30Weisgraber K.H. Innerarity T.L. Mahley R.W. J. Biol. Chem. 1982; 257: 2518-2521Abstract Full Text PDF PubMed Google Scholar). However, recent studies showing apoE immunoreactivity in the cytoplasm of neurons (31Han S.-H. Einstein G. Weisgraber K.H. Strittmatter W.J. Saunders A.M. Pericak-Vance M. Roses A.D. Schmechel D.E. J. Neuropathol. Exp. Neurol. 1994; 53: 535-544Crossref PubMed Scopus (183) Google Scholar) suggest differences between neuronal and nonneuronal cells in the metabolism of apoE.We first examined the ability of β-VLDL together with either apoE3 or apoE4 to deliver lipids to the cells. Incubation of the cells with β-VLDL and apoE3 or apoE4 for 48 h did in fact lead to similar levels of lipid accumulation (apoE3 +β-VLDL, 115 ± 3.9 μg cholesterol/mg of cell protein; apoE4 +β-VLDL, 121 ± 5.8 μg cholesterol/mg of cell protein), suggesting that the stimulation of β-VLDL uptake was similar with the two apoE isoforms. In comparison, Neuro-2a cells accumulated 20 ± 0.9 and 86 ± 2.6 μg of cholesterol/mg of cell protein when incubated with N2 medium and β-VLDL alone, respectively. Examination of the uptake of fluorescently labeled β-VLDL in the presence of either apoE3 or apoE4 supported the conclusion that apoE3 and apoE4 stimulated β-VLDL uptake similarly.To determine whether apoE3 and apoE4 were processed differently by neurons and by fibroblasts, we incubated Neuro-2a cells or murine fibroblasts with apoE3 or apoE4 in the presence of β-VLDL and examined the accumulation of apoE in the cells. Immunocytochemical detection of apoE in Neuro-2a cells incubated with β-VLDL and either apoE3 or apoE4 revealed that both isoforms were present within neurons (Fig. 3). There was, however, a substantial difference in the intensity of reactivity of apoE3 (Fig. 3A) and apoE4 (Fig. 3B). Apolipoprotein E3 was present both in the cell body and in the neurites at a substantially higher concentration than was apoE4 (Fig. 3, A and B). Both apoE3 and apoE4 were observed in nearly all serial optical sections made throughout the cell, suggesting that apoE was intracellular (Fig. 3, A and B).Figure 3:Immunocytochemical localization of apoE in Neuro-2a cells. Neuro-2a cells were grown for 2 days in medium containing β-VLDL (40 μg of cholesterol/ml) together with 30 μg/ml of either human apoE3 (A) or human apoE4 (B). Immunocytochemistry was performed as described under “Experimental Procedures.” Serial optical sections (∼1 μm in thickness) were made from the top (section 1) to the bottom (section 6) of the cells using a confocal laser scanning microscope. Scale bar = 15 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The possibility that apoE was localized intracellularly was examined using two additional approaches. First, Neuro-2a cells were incubated with β-VLDL and either apoE3 or apoE4 and treated with suramin (a polyanion known to remove lipoproteins nonspecifically bound to the cell surface and specifically bound to their receptors), followed by immunocytochemistry for apoE. Treatment with suramin did not significantly reduce the apoE immunoreactivity associated with the cells. Second, immunocytochemical studies were performed in cells that were not permeabilized to permit access of antibody to the cytoplasm. No immunoreactivity of apoE was observed in nonpermeabilized cells incubated with β-VLDL together with either apoE3 or apoE4. These results demonstrated that the apoE was intracellular. The observed intracellular accumulation of apoE was unexpected, since lipoproteins and their apoproteins, when internalized by nonneuronal cell types, are rapidly degraded. To determine if the accumulation of apoE was specific to neurons, we performed similar immunocytochemistry experiments in murine fibroblasts. In these studies no apoE immunoreactivity was observed in the fibroblasts incubated with β-VLDL and either apoE3 or apoE4, suggesting either that apoE enters neurons and fibroblasts through different pathways or that in neurons apoE, especially apoE3, can escape lysosomal degradation. These results demonstrate that apoE3 is retained in Neuro-2a cells to a greater extent than apoE4 and that the metabolism of apoE in neuronal and nonneuronal cells is different.The differences observed in the accumulation of apoE3 and apoE4 were confirmed by incubating the neurons with 125I-labeled apoE and β-VLDL at 37°C for 48 h, and the amount of cell-associated apoE (bound and internalized) was quantitated. A differential accumulation of 125I-apoE was observed, with twice as much 125I-apoE3 as 125I-apoE4 being associated with the cells at the end of the incubation period (Fig. 4).Figure 4:Cell association of 125 I-apoE with Neuro-2a cells. Neuro-2a cells were grown for 2 days in medium containing β-VLDL (40 μg of cholesterol/ml) together with 30 μg/ml of either 125 I-apoE3 or 125 I-apoE4. Following incubation, the radioactivity associated with the cells (representing b" @default.
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• W2158965128 date "1995-08-01" @default.
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• W2158965128 title "The Inhibitory Effect of Apolipoprotein E4 on Neurite Outgrowth Is Associated with Microtubule Depolymerization" @default.
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