A Minimally Lipidated Form of Cell-derived Apolipoprotein E Exhibits Isoform-specific Stimulation of Neurite Outgrowth in the Absence of Exogenous Lipids or Lipoproteins*

Ronald B. DeMattosDagger , Linda K. Curtiss§, and David L. WilliamsDagger

From the Dagger  Department of Pharmacological Sciences, University Medical Center, State University of New York, Stony Brook, New York 11794 and the § Departments of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, California 92037

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Within the central nervous system, apolipoprotein E (apoE) synthesis is increased in response to nerve injury, a finding that may reflect a role for apoE in neuronal remodeling. Recent studies show that apoE3 promotes and apoE4 inhibits neurite outgrowth in cultured neuronal cells. Interestingly, these isoform-specific effects are observed only when apoE is presented to cells in the presence of an exogenous lipid source such as rabbit beta -very low density lipoprotein (beta -VLDL), making it difficult to discern the biologically active form of apoE or to understand the role of the lipid source. In the present study we tested whether a cell-derived lipidated form of apoE can alter neurite outgrowth in the absence of beta -VLDL by constructing Neuro-2a cell lines expressing high levels of apoE. Our results showed that endogenous apoE3 stimulated neurite outgrowth, whereas the endogenous apoE4 isoform was neutral. Furthermore, beta -VLDL antagonized the stimulatory effects of the endogenous apoE3. Characterization of the secreted apoE3 indicated that the neurite outgrowth-stimulating activity could be recovered from culture medium with an anti-apoE immunoaffinity column and was present in a poorly lipidated particle with a density between 1.19 and 1.26 g/ml. These results indicated that the biological activity of apoE3 in stimulating neurite outgrowth was inherent in the cell-derived apoE particle and was not dependent on either (a) an interaction of apoE3 with an artificial lipid source or (b) independent actions of apoE3 and beta -VLDL.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Apolipoprotein E (apoE)1 is a 299-amino acid glycoprotein (1) first identified in 1973 as a constituent of human very low density lipoprotein (VLDL) and subsequently found in all lipoprotein classes. ApoE plays a key role in cholesterol homeostasis by mediating the hepatic clearance of plasma cholesteryl ester-rich VLDL and chylomicron remnants. In contrast to other apolipoproteins, which are synthesized only in liver and gut, apoE is expressed in a variety of tissues and cell types including abundant expression by steroidogenic cells and astrocytes of the brain (1-4). ApoE is present in cerebrospinal fluid (CSF) at 5-10 µg/ml, which is approximately 5-10% of its plasma concentration (5, 6). The endogenous lipoproteins in CSF are predominantly HDL-like particles that arise from astrocyte-derived apoE in addition to apoAI, which presumably crosses the blood-brain barrier (7, 8). The role of brain apoE is not known, although its expression appears to be correlated with nerve injury and neuronal remodeling (8-13).

The three common isoforms of human apoE result from cysteine-arginine interchanges at residues 112 and 158 (14). ApoE3 contains cysteine at residue 112 and arginine at residue 158. ApoE2 contains 2 cysteines, whereas apoE4 contains arginines at these residues. A correlation between the apoE epsilon 4 allele and late-onset familial Alzheimer's disease has been identified (15-17). However, the role of apoE in the pathology of Alzheimer's disease is still unknown. There is evidence that apoE interacts with protein components of both pathologic features of Alzheimer's disease (amyloid plaques and neurofibrillary tangles) in an isoform- specific manner (18, 19). In addition, purified delipidated apoE will stimulate isoform-specific differences in neurite outgrowth when added to neuronal cells in culture (14, 20, 21). A murine neuroblastoma cell line, Neuro-2a, exposed to delipidated apoE3 has significantly longer neurite extensions when compared with either control cells or cells treated with apoE4 (14). The delipidated apoE4 isoform has an inhibitory effect on neurite outgrowth that correlates with destabilization of microtubular arrays (14). Interestingly, these isoform-specific differences are only observed when an exogenous lipid source (rabbit beta -VLDL) is added along with the delipidated apoE isoforms. Neither the rabbit beta -VLDL, which contains abundant rabbit apoE, nor the human apoE alone alters neurite outgrowth.

Neuro-2a cell lines expressing low quantities of apoE isoforms display similar isoform-specific differences in the presence of rabbit beta -VLDL, yet are indistinguishable from control cells that lack apoE expression in the absence of the exogenous lipid source (22). Multiple lipid sources (VLDL, triglyceride-rich emulsions, HDL, and HDL-like CSF lipoproteins) can substitute for beta -VLDL to propagate similar isoform-specific differences (21-23). However, a puzzling aspect of these studies is that CSF lipoproteins fail to induce a difference in neuritogenesis unless they are enriched with exogenous human apoE (23), even though the CSF HDL-like particles already contain apoE (7, 23). Importantly, these studies do not identify a physiologically significant or biologically active form of apoE because they require artificial lipid sources to observe apoE-induced stimulated neurite outgrowth.

If apoE-mediated alterations in neurite outgrowth are relevant to neuronal remodeling events in vivo, it is likely that the biologically active form of apoE is a cell-derived, lipidated complex that can act in the absence of beta -VLDL or other artificial lipid sources. To test this possibility we constructed Neuro-2a cell lines that secrete higher levels of the apoE isoforms. These cells produced a minimally lipidated apoE3 that stimulated neurite outgrowth in the absence of exogenous lipid. Under identical conditions, apoE4 had no effect on neurite outgrowth, indicating that the E4 isoform was neutral and not inhibitory. Furthermore, exogenous beta -VLDL antagonized the stimulatory effects of cell-derived apoE3. Thus, these studies identified a minimally lipidated form of apoE that was biologically active in promoting neurite outgrowth.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Apolipoprotein E Expression Vectors

A high expression apoE3 vector was constructed by inserting a PCR-generated apoE3 cDNA into pcDNA3 (Invitrogen). The apoE3 insert was produced with the following PCR conditions: 17 ng of template (pHE54, generous gift of John Taylor and David Walker, Gladstone Institute of Cardiovascular Disease), 5 units of Taq polymerase, 50 µM dNTPs, 1.5 mM MgCl, 0.01% gelatin, 10% Me2SO, 50 mM KCl, 10 mM Tris-HCl, pH 8.3, and 5 µM high annealing temperature primers (each incorporating a unique 5' restriction enzyme site). The translational enhancer of the alpha mosaic virus from the expression plasmid pCMV4 (24) was inserted 4 bases upstream of the apoE3 cDNA to yield pC1E3. The apoE4 expression vector, pC1E4, was made by substituting the SacII-FseI fragment of pC1E3 with the corresponding fragment from the apoE4 sequence in plasmid pFE (25). All PCR inserts and junctions were verified by standard sequencing techniques.

Production of Stable ApoE3- or ApoE4-transfected Neuro-2a Cell Lines

Neuro-2a cells were maintained in a 37 °C humidified 95% air, 5% CO2 incubator in medium A (Dulbecco's modified Eagle's medium/Ham's F12 (1:1) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Atlanta Biologicals), 4 mM glutamine, 100 units/ml penicillin, 100 units/ml streptomycin sulfate, and 0.25 µg/ml amphotericin B). Neuro-2a cells were plated at 1.0 × 106 cells in 10 ml of medium A per 10-cm dish and were transfected with 20 µg of plasmid using a standard calcium phosphate precipitation protocol (26). Stable integrants were selected and maintained in medium B (medium A plus 350 µg/ml G418 (Life Technologies, Inc.)).

Characterization of Parental, ApoE3, and ApoE4 Neuro-2a Cell Lines

Northern Blotting Analysis-- Total RNA was isolated from confluent 10-cm plates of parental, apoE3, and apoE4 cell lines using Stat-60 (Tel-Test) reagent and protocol. Total RNA (25 µg) was denatured, subjected to electrophoresis through a 1.2% agarose gel containing 2.2 M formaldehyde, and transferred to a nylon membrane by overnight capillary transfer. The membrane was probed with a 32P random-primed apoE fragment (nucleotides 209-653), and hybridization was visualized with a Molecular Dynamics PhosphorImager.

Western Blotting Analysis-- Cells were plated at 1.0 × 107 cells in 10 ml of medium A per 10-cm dish, incubated overnight at 37 °C, washed twice with medium A, and incubated for an additional 24 h in 10 ml of fresh medium A. Conditioned medium was removed; cells were washed 2 times with PBS and solubilized in 0.5 ml of 2% SDS in PBS for total protein determination. Conditioned medium was run on a 10% SDS-polyacrylamide gel, electrophoretically transferred to nitrocellulose, and blocked for 1 h at room temperature in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl (TBS) containing 7% nonfat milk, and 0.05% Tween 20. The blocked membrane was incubated with affinity purified polyclonal goat anti-human apoE antibody (Biodesign International) at 2 µg/ml overnight at room temperature in TBS containing 1% nonfat milk and 0.2% Tween 20. The membrane was washed three times with TBS containing 0.05% Tween 20 and incubated with a horseradish peroxidase-conjugated anti-goat IgG (Sigma) for 1 h at room temperature in TBS containing 1% nonfat milk and 0.05% Tween 20. Bands were visualized by enhanced chemiluminescence (Amersham Corp.).

Immunocytochemistry

Neuro-2a cells were plated onto 18-mm glass circle coverslips in 12-well tissue culture plates at 5.0 × 103 cells in 4 ml of medium A per well and allowed to adhere overnight at 37 °C. Cells were washed twice with PBS and fixed in ice-cold PBS containing 3% paraformaldehyde for 30 min. Cells were washed twice with PBS, permeabilized in ice-cold PBS containing 0.5% Triton X-100 for 15 min, and blocked in PBS containing 10% FBS and 0.5% Triton X-100 for an additional 15 min at 37 °C. Cells were incubated with an affinity purified polyclonal goat anti-human apoE antibody at 2 µg/ml in PBS containing 1% FBS and 0.5% Triton X-100 for 1 h at 37 °C. The cells were washed three times with PBS containing 0.5% Triton X-100 and incubated with a rhodamine-conjugated donkey anti-goat IgG antibody (Jackson Immuno Research) at 1:2000 in in PBS containing 1% FBS and 0.5% Triton X-100 for 1 h at 37 °C. Glass slips were washed three times with PBS containing 0.5% Triton X-100, mounted in SlowFade (Molecular Probes), and analyzed with a Bio-Rad MRC-600 scanning confocal system mounted on a Nikon Diaphot inverted microscope. Images were saved as Bio-Rad Pic files and were subsequently converted into Tif files.

ELISA Analysis

ApoE concentration in conditioned medium was determined by ELISA as follows: 96-well plates were coated with 100 µl of affinity purified goat anti-human apoE antibody (5 µg/ml) in PBS overnight at 4 °C. The plate was washed twice with PBS and blocked with 400 µl of PBS containing 7% non-fat milk for 1 h at 37 °C and then washed twice. The coated plate was incubated at 37 °C for 1 h with 100 µl of conditioned medium and washed twice, and 100 µl of a biotin-labeled affinity purified goat anti-human apoE polyclonal diluted 1:1000 in PBS containing 0.5% non-fat milk was added and incubated at 37 °C for 30 min. After washing four times, 100 µl of streptavidin/horseradish peroxidase (Life Technologies, Inc.) diluted 1:1000 in PBS containing 0.5% non-fat milk was added, and the plate was incubated at 37 °C for 30 min. The plate was washed six times; 150 µl of 3,3',5,5'-tetramethylbenzidine liquid substrate (Sigma) was added, and color development was monitored at 650 nm. A standard curve was generated with purified human apoE (Panvera) at concentrations ranging from 1 to 50 ng. Samples were assayed in triplicate.

Neurite Extension Assay

Parental, apoE3, and apoE4 Neuro-2a cell lines were trypsinized and plated at low cell densities on days -4 and -2. At time 0 cells were trypsinized for exactly 2 min and subsequently plated in medium A at a density of 2.0 × 104 cells per 60-mm dish. After 2 h at 37 °C, medium was removed, the dish washed twice with basal medium (medium A lacking FBS), and freshly made basal medium plus N2 growth supplement (Life Technologies, Inc.) either with or without rabbit beta -VLDL (40 µg of cholesterol/ml) was added. Cells were incubated for 96 h at 37 °C with 1 medium change at 48 h. At 96 h, cells were fixed in PBS containing 2.5% glutaraldehyde for 30 min at room temperature and nonspecifically stained with 0.002% acridine orange in PBS for 30 min. Cells were washed three times with PBS and coverslipped. Images were captured with a 20 × objective and filter group specific for rhodamine fluorescence on the same system stated above. Images were converted into TIF format and analyzed with the UTHSCSA Image Tool program (developed at the University of Texas Health Science Center, San Antonio, TX).2 Every cell that contained a process longer than the cell diameter was measured (longest neurite only). Initial samples contained microsphere calibration standards (Duke Scientific Corp.) of a mean diameter of 9.975 ± 0.061 µm.

Affinity Purification of Apolipoprotein E Lipid Particles

An affinity chromatography isolation procedure utilizing the human apoE-specific monoclonal antibody 1E was employed. Purified 1E antibody was coupled to CNBr-activated Sepharose 4B (Sigma) by incubating 10 mg of antibody in 0.1 M NaHCO3, pH 8.3, 0.5 M NaCl with 2 ml of pre-swelled CNBr-activated Sepharose 4B on a rotary shaker overnight at 4 °C. The gel was washed with 10 ml of 0.1 M NaHCO3, pH 8.3, 0.5 M NaCl, and remaining active groups were blocked for 2 h with 1 M ethanolamine, pH 8.0. The gel was then washed with three cycles of alternating pH using 10 ml of 0.1 M acetate, pH 4.0, 0.5 M NaCl, and 0.1 M Tris-HCl, pH 8.0, 0.5 M NaCl. The coupled gel was packed into a glass column, and conditioned media from cells secreting apoE3 were recycled at ~1 ml/min overnight at 4 °C. The column was washed with 100 ml of PBS, and apoE3-containing particles were eluted in 60 ml of 100 mM triethylamine, pH 11.5, and immediately neutralized with 20 ml of 1 M sodium phosphate, pH 6.8. The apoE3 particles were dialyzed and concentrated into PBS and subsequently into basal medium using Centriprep 50 concentrators (Amicon). The final apoE3 preparation was sterilized by filtration through a pre-blocked (1% bovine serum albumin in PBS) 0.2-µm filter (Schleicher & Schuell). Each apoE3 preparation was analyzed by Western blotting and quantified by ELISA as described above.

Trans-addition Analysis of Affinity Purified ApoE Particles

Parental Neuro-2a cells were plated at a density of 2.0 × 103 cells per well of a 24-well tissue culture plate (Costar) in medium A. After 2 h at 37 °C, medium was removed; the dish was washed twice with basal medium (medium A lacking FBS), and incubation was continued for 48 h at 37 °C with either basal medium plus N2 supplement alone or with immunopurified apoE3 (30 µg/ml) in basal medium plus N2 supplements. The cells were fixed, stained, and analyzed as stated above. All microscopy experiments were coded before confocal image analysis and again before neurite measurements using double blind coding procedures.

Lipoproteins

Preparation of the rabbit beta -VLDL was as described (27), except all protease inhibitors were omitted. Rabbit beta -VLDL was stored at 4 °C under nitrogen and was used within 2 weeks of isolation. HDL3 (1.125 g/ml < rho  < 1.225 g/ml) was isolated by standard ultracentrifugation techniques (28). The lipidation state of apoE secreted by Neuro-2a cells was determined by ultracentrifugation using KBr for density adjustment. Conditioned medium (4 ml) was adjusted to rho  = 1.20 g/ml, underlaid with 4 ml at rho  = 1.34 g/ml, and overlaid with 4 ml at rho  = 1 .10 g/ml. Gradients were centrifuged at 38,000 rpm in an SW41 rotor for 48 h at 18 °C. Fractions were collected from the top of the tube, density measured by weight, and apoE content determined by ELISA. Secreted apoE was delipidated with diethyl ether/ethanol as described (29).

Miscellaneous

Protein was measured with an IgG standard (30). Statistical significance was determined by Student's t test.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

High capacity expression vectors for stable expression of human apoE were constructed by incorporating the alpha mosaic virus translational enhancer into the apoE 5'-untranslated region within the pcDNA3 vector in which expression was driven by the strong cytomegalovirus promoter/enhancer (Invitrogen). Multiple cell lines for each apoE isoform were isolated and characterized for apoE production. The Northern blot in Fig. 1 shows similar levels of apoE3 and apoE4 mRNAs in representative cell lines expressing the transfected apoE cDNA, whereas apoE mRNA was not detected in the parental cell line. Conditioned media from each of these cell lines were analyzed by Western blotting using an affinity purified apoE polyclonal antibody as shown in Fig. 2. The parent Neuro-2a cell line was completely devoid of apoE immunoreactivity, whereas the apoE3 and apoE4 cell lines each showed the expected 36-kDa apoE band. Quantification of apoE accumulation in conditioned medium showed high levels of expression for both apoE3 and apoE4 cell lines (Table I). These values were approximately 100-fold greater than previously reported for apoE-expressing Neuro-2a cell lines (22).


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Fig. 1.   Northern blotting of apoE mRNA in stably transfected cell lines. Total RNA (25 µg/lane) prepared from the parental Neuro-2a line and from apoE3-expressing and apoE4-expressing Neuro-2a cell lines was denatured and separated by electrophoresis on a 1.2% agarose gel. After transfer to a nylon membrane, the RNA was visualized by ethidium bromide staining to verify that equal amounts of intact RNA were present (A). The membrane was probed with a 32P random primed apoE fragment (nucleotides 209-653), and hybridization was visualized with a Molecular Dynamics PhosphorImager (B).


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Fig. 2.   Western blotting of apoE secreted by stably transfected cell lines. Conditioned media (100 µg of protein) were separated by SDS-10% polyacrylamide gel electrophoresis and were electroblotted to a nitrocellulose membrane. The membrane was probed with an affinity purified polyclonal goat anti-human apoE antibody. After incubation with a horseradish peroxidase-conjugated anti-goat IgG, bands were visualized by enhanced chemiluminescence. The slight difference in mobility of the apoE3 and apoE4 bands is due to curvature in the running front and was not seen in other experiments.

                              
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Table I
Apolipoprotein E secretion
ApoE concentration was measured for two clones expressing each apoE isoform and the parental Neuro-2a cells. Each cell line was plated at 2.0 × 104 cells per 60-mm dish in medium A and incubated at 37 °C for 2 h. The plates were washed with basal medium; 4 ml of medium B (basal + N2 supplements) were added, and the plates were incubated at 37 °C for 48 h. Triplicate plates for each cell line were analyzed for apoE accumulation in the medium by ELISA, and total cell protein was determined at 48 h. Triplicate plates for each cell line also were allowed to continue another 48 h in fresh medium B prior to measurements of apoE and total cell protein. The apoE values are means ± standard deviation. Neurite extensions were measured for each cell line as described in the legend to Fig. 5. ND, none detected.

Immunocytochemical localization of the Neuro-2a-associated apoE showed a punctate staining pattern throughout the entire cell with the exception of the nucleus that was devoid of staining (Fig. 3, C and D). ApoE staining was particularly concentrated within the growth cones of the extending neurites in both apoE3- and apoE4-expressing cells. Each of the above analyses indicated no detectable apoE expression by the parental cell line and, importantly, similar levels of apoE expression by apoE3 and apoE4 cell lines.


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Fig. 3.   Immunocytochemical detection of apoE in stably transfected cell lines. Neuro-2a parental cells (A and B), Neuro-2a apoE3-secreting cells (C), and Neuro-2a apoE4-secreting cells (D) were grown on glass coverslips. ApoE localization was detected with an affinity purified polyclonal goat anti-human apoE antibody and a rhodamine-conjugated secondary antibody (A, C, and D). The white arrows highlight the concentrated localization of apoE in the growth cone domains in the apoE isoform-expressing Neuro-2a cells. B is the transmitted light image of the parental Neuro-2a cells found in A (black arrows highlight 2 Neuro-2a cells). All images were collected by confocal microscopy with a 40 × oil immersion lens.

The above cell lines were analyzed in a neurite extension assay. Fig. 4A shows representative confocal images from the three cell lines at the conclusion of the 96-h assay in the absence of beta -VLDL. Strikingly, the cells secreting apoE3 had substantially longer neurite extensions than either the apoE4 or control cells. To quantify these differences, multiple images were saved from three independent experiments and analyzed for neurite length. Fig. 5A demonstrates that the apoE3-secreting cell line had significantly longer neurites (214%, p = 0.0001 versus control) than either control (set to 100%) or apoE4-secreting cells (114%) in the absence of beta -VLDL. The data were further analyzed to ascertain whether the observed mean differences were indicative of uniform behavior within the cell population or due to a minor subpopulation of cells with dramatically longer neurites. Fig. 5B illustrates that the distribution of neurite lengths was shifted throughout the cell population in the apoE3-secreting cells, whereas the apoE4-secreting cells were nearly indistinguishable from the control cells.


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Fig. 4.   Cell-derived apoE isoform effects on neurite outgrowth in the absence and presence of beta -VLDL. Cells were grown for 96 h in N2-supplemented basal medium alone (A) or in the presence of beta -VLDL (40 µg of cholesterol/ml) (B). Cells were fixed, permeabilized, and nonspecifically stained with acridine orange at the conclusion of the 96-h assay. Confocal images were captured using a 20 × objective and filter group specific for rhodamine fluorescence. The figure shows the negative images transformed by the Image Tool program.


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Fig. 5.   Quantification of cell-derived apoE isoform effects on neurite outgrowth in the absence and presence of beta -VLDL. Cells were grown for 96 h in N2-supplemented basal medium alone or in the presence of beta -VLDL (40 µg of cholesterol/ml). Cells were fixed, permeabilized, and nonspecifically stained with acridine orange. Multiple confocal images for each treatment in all three cell lines were captured using a 20 × objective and analyzed in the Image Tool program. Neurite extensions were determined by measuring the longest neurite outgrowth from 60 to 90 responsive cells (cells containing a neurite that is at least 1 cell diameter in length). A shows the average neurite extensions from three independent experiments both in the absence and presence of beta -VLDL (error bars indicate standard error). The level of significance of the differences was calculated for the apoE3-secreting cells compared with control Neuro-2a cells in the absence of beta -VLDL (*, p = 0.0001) and for the apoE3-secreting cells in the absence of beta -VLDL compared with apoE3-secreting cells in the presence of beta -VLDL (**, p = 0.0005). These data were analyzed to determine the percentage of total cells (N) expressing neurites of defined lengths in the absence of beta -VLDL (B) or in the presence of beta -VLDL (C). Error bars in B and C indicate the standard deviation. Error bars not shown are within the area of the symbol.

The apoE concentration in the medium after 48 and 96 h of the extension assay was measured in two clonal lines expressing each apoE isoform. Table I shows some variation in secreted apoE, yet the neurite extension results for these clones were consistent with isoform-specific modulation that was relatively unaffected by the small differences in secreted apoE. For example, the 1C and 1E4C clones showed identical apoE concentrations at 48 h, and the 1E4C apoE concentration differed from the 1C concentration only by 20% at 96 h. Nevertheless, the apoE3-expressing 1C clone showed a stimulation of neurite outgrowth (p = 0.0001), whereas the apoE4-expressing 1E4C clone did not. These data indicate that the cell-derived apoE3 stimulated neurite outgrowth in the absence of beta -VLDL, whereas under identical conditions, apoE4 was neutral.

Fig. 4B shows images of the same cell lines after a 96-h exposure to beta -VLDL. Surprisingly, the addition of beta -VLDL to the extension assays reduced apoE3 neurite extensions compared with the apoE3 extensions in the absence beta -VLDL. As shown in Fig. 5A, beta -VLDL reduced neurite outgrowth in apoE3-expressing cells from 214% of control to 128% of control (p = 0.0005), whereas beta -VLDL had no significant effect (p > 0.05) on neurite outgrowth in the control Neruo-2a and apoE4-expressing cell lines. As shown in Fig. 5C, beta -VLDL appeared to normalize the neurite length distribution for the three cell lines. Thus, beta -VLDL inhibited neurite outgrowth in the apoE3-expressing cells (p = 0.0005) but had no effect on control or apoE4-expressing cells (p > 0.05).

An important question from these results was whether the effect of apoE3 on neurite outgrowth reflected an extracellular action of the secreted protein. Alternative possibilities were that the effect was due to intracellular apoE or was an indirect result of high level apolipoprotein secretion which might perturb lipid homeostasis within the cell. To test these possibilities, we isolated the secreted apoE3 from conditioned medium by affinity chromatography with monoclonal antibody 1E. A series of five trans-addition experiments were performed in which the isolated apoE3 (30 µg/ml) was incubated with parental Neuro-2a cells in a 48-h extension assay. Fig. 6A illustrates that the immunopurified apoE3 maintained its ability to stimulate a significant increase in the average neurite length (154% of control, p = 0.0002). Furthermore, the population distribution of neurite lengths for the cells treated with the purified apoE3 closely resembled the distribution seen in the transfected apoE3 cell line in the absence of beta -VLDL (Fig. 6B).


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Fig. 6.   Effect of immunopurified apoE3 on neurite outgrowth in Neuro-2a cells. Neuro-2a cells were incubated with N2-supplemented basal medium alone or in the presence of 30 µg/ml cell-derived apoE3 for 48 h. Cells were fixed, permeabilized, and nonspecifically stained with acridine orange. Multiple confocal images for either treatment were captured using a 20 × objective and analyzed with the Image Tool program. Average neurite extensions were determined by measuring the longest neurite from 30 to 80 responsive cells in each of five independent experiments (A). The error bars indicate the standard error. The level of significance of the difference was p = 0.0002. These data were analyzed to determine the percentage of Neuro-2a cells (N) expressing neurites of defined lengths (B). Error bars indicate the standard deviation.

Previous studies have shown that apoE secreted by non-hepatic cells is poorly lipidated (31, 32). Therefore, the lipidation state of apoE secreted by Neuro-2a cells was determined by density gradient ultracentrifugation. Both secreted apoE isoforms floated at a density of 1.19 to 1.26 g/ml, indicating a poorly lipidated particle (Fig. 7, A and B). None of the secreted apoE had a density typical of apoE-containing plasma HDL (Fig. 7C), and secreted apoE was shifted to higher density upon delipidation (Fig. 7D). These results indicate that a physiologically relevant and lipid-poor form of secreted apoE3 enhanced neurite outgrowth.


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Fig. 7.   Density distribution of Neuro-2a-derived apoE. Cell-derived apoE isoforms were analyzed for density distribution by density gradient ultracentrifugation. Conditioned media from apoE3- and apoE4-expressing Neuro-2a cells were centrifuged in KBr density gradients for 48 h, and fractions were taken from the top of the tube. Fractions were assayed by ELISA for apoE content, and densities were determined by weight. The graphs show the percent of total apoE3 (A), apoE4 (B), apoE-HDL3 (C), or delipidated apoE3 in comparison to apoE3 in conditioned medium (D) per fraction.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

We produced high expressing apoE3 and apoE4 Neuro-2a cell lines to test whether cell-derived apoE could exhibit isoform-specific stimulation of neurite outgrowth in the absence of an exogenous lipid source. ApoE3 stimulated neurite outgrowth in the absence of beta -VLDL or other exogenous lipid sources. Furthermore, cell-derived lipidated apoE3 isolated by immunoaffinity chromatography was active in promoting neurite outgrowth in parental Neuro-2a cells. Nathan et al. (14) discovered that the co-addition of purified delipidated apoE and beta -VLDL to Neuro-2a cells elicits isoform-specific differences in neurite outgrowth, effects also seen in other cell lines and primary neuronal cultures (20-23, 33). Interestingly, these isoform-specific effects could not be duplicated by apoE addition alone. Similarly, Neuro-2a cell lines that express low amounts of apoE isoforms also required beta -VLDL to elicit effects on neurite outgrowth (22). It was unclear from these studies whether apoE3 stimulates neurite outgrowth independently but in concert with an exogenous lipid or whether an active form of apoE was formed only when it was combined with the exogenous lipid source. Our results indicated that the biological activity of apoE3 in stimulating neurite outgrowths was inherent in the cell-derived lipidated particle and was not dependent on either (a) an interaction of apoE3 with an artificial lipid source or (b) independent actions of apoE3 and beta -VLDL on Neuro-2a cells.

We demonstrated that cell-derived apoE4 was neutral when challenged in a neurite extension assay in the absence of beta -VLDL. Although some studies have implicated apoE4 as being inhibitory to neurite outgrowth in the presence of an artificial lipid source (14, 22), other studies support the conclusion that apoE4 is neutral. Holtzman et al. (21) showed an apoE3 enhancement of neurite outgrowth in the neuronal cell line GT1-1 trk9 only in the presence of beta -VLDL and nerve growth factor but no significant difference between apoE4-treated cells and controls under the same conditions. Experiments with GT1-1 trk9 cells give similar results when HDL-sized particles from human CSF are substituted for beta -VLDL (23). In further studies, Puttfarcken et al. (34) showed that bulk conditioned medium from HEK-293 cells stably expressing either apoE3 or apoE4 isoforms are moderately stimulatory in primary hippocampal cultures. These experiments (21, 34) as well as the present results argue that the apoE4 isoform is devoid of neurite-stimulating activity but is not inhibitory when presented to cells alone, in combination with exogenous lipids, or as a cell-derived lipidated particle.

When present in the neurite extension assay, beta -VLDL antagonized the stimulatory effect of apoE3, an effect that was not detected in previous studies in which beta -VLDL was required for the effect of delipidated apoE (14, 20-23, 33). It may be that in experiments with delipidated apoE, beta -VLDL acts as a lipid source to facilitate formation of an active apoE particle and at the same time antagonizes the action of the lipidated apoE. In this case, the level of apoE3-stimulated neurite outgrowth would depend on the balance between these two factors. Furthermore, it is unlikely that the antagonism observed in the present study is a nonspecific cytotoxic effect because beta -VLDL alone modestly stimulated neurite outgrowth (111%), an effect also observed by others (14, 20, 22). The antagonism by beta -VLDL may reflect sequestration of secreted apoE or competition at a cellular receptor site.

Cell-derived apoE occurred as a poorly lipidated particle with a density of 1.19 to 1.26 g/ml. Because this minimally lipidated form of apoE was biologically active in the absence of an exogenous lipid source, it was unlikely that the neurite outgrowth stimulation reflects an action of apoE to simply deliver lipid to the cell for membrane formation. We speculate that apoE acted extracellularly to direct neurite extension or as a paracrine factor to stimulate neurite growth.

How does the lipidated apoE3 particle stimulate neurite outgrowth although apoE4 remains neutral? The LDL receptor and the LDL receptor-related protein (LRP) bind apoE containing lipoproteins and are expressed by neuronal cells (35-37). Evidence from several studies implicate LRP in the apoE isoform-specific stimulation of neurite outgrowth (21, 22, 38). In particular, inhibition of the apoE3 effect on neurite outgrowth by anti-LRP antibodies provides strong evidence for the involvement of LRP. However, binding studies with the LRP show no isoform-specific differences between apoE3 and apoE4 (27). This discrepancy may be resolved by recent reports of other apoE receptors. A new apoE receptor has been described which localizes to neurons and has a high homology to the LDL receptor (39). Additional brain-specific receptors with high homology to the LRP (40) and the LDL receptor (41) also have been identified. The present findings that a cell-derived form of apoE3 is active in stimulating neurite outgrowth will permit both physical characterization of the particles and analysis of isoform-specific interactions with these recently described neuronal receptors.

    ACKNOWLEDGEMENTS

We thank Miguel Berrios and William Theurkauf for advice on confocal microscopy and David Colflesh, Katherine Richards, and Anna Demian for technical assistance.

    FOOTNOTES

* This work was supported by Grants HL 32868 and HL 35297 from the NHLBI of the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacological Sciences, University Medical Center, State University of New York, Stony Brook, NY 11794. Tel.: 516-444-3083; Fax: 516-444-3218; E-mail: Dave{at}Pharm.som.sunysb.edu.

1 The abbreviations used are: apoE, apolipoprotein E; VLDL, very low density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein; CSF, cerebrospinal fluid; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; ELISA, enzyme-linked immunosorbent assay; LRP, LDL receptor-related protein; FBS, fetal bovine serum; PCR, polymerase chain reaction.

2 Available from the internet by anonymous FTP from ftp:maxrad6.uthscsa.edu.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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