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Endocrinology, doi:10.1210/en.2003-1707
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Endocrinology Vol. 145, No. 7 3065-3073
Copyright © 2004 by The Endocrine Society

Estrogen Facilitates Neurite Extension via Apolipoprotein E in Cultured Adult Mouse Cortical Neurons

Britto P. Nathan, Anna G. Barsukova, Fei Shen, Mary McAsey and Robert G. Struble

Department of Biological Sciences (B.P.N., A.G.B., F.S.), Eastern Illinois University, Charleston, Illinois 61920; and Department of Obstetrics and Gynecology (M.M.), and Center for Alzheimer Disease (R.G.S.), Southern Illinois University School of Medicine, Springfield, Illinois 62794

Address all correspondence and requests for reprints to: Robert G. Struble, Ph.D., P.O. Box 19628, Center For Alzheimer Disease and Related Disorders, Southern Illinois University School of Medicine, Springfield, Illinois 62794-9628. E-mail: Rstruble{at}siumed.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Literature review suggests a close relationship between estrogen and apolipoprotein E (ApoE) in the central nervous system. Epidemiology studies show that estrogen replacement therapy (ERT) decreases the morbidity from several chronic neurological diseases. Alleles of ApoE modify the risk for and progression of the same diseases. ApoE levels in the rodent brain vary during the estrous cycle and increase after 17ß-estradiol administration. Both estradiol and ApoE3, the most common isoform of human ApoE, increase the extent of neurite outgrowth in culture. Combined, these observations suggest a common mechanism whereby estrogen may increase ApoE levels to facilitate neurite growth. We tested this hypothesis by characterizing the effects of estradiol and ApoE isoforms on neurite outgrowth in cultured adult mouse cortical neurons. Estradiol increased ApoE levels and neurite outgrowth. ApoE2 increased neurite length more so than ApoE3 in the presence of estradiol. Estradiol had no effect on neurite outgrowth from mice lacking the ApoE gene or when only ApoE4, the isoform of ApoE that is associated with increased risk of neurological disease, was exogenously supplied. Cultures from mice transgenic for human ApoE3 or ApoE4 showed the same isoform-specific effect. Neuronal internalization of recombinant human ApoE3 was greater than ApoE4, and ApoE3 was more effective than ApoE4 in facilitating neuronal uptake of a fatty acid. We conclude that estradiol facilitates neurite growth through an ApoE-dependent mechanism. The effects of ERT on chronic neurological diseases may vary with ApoE genotype. The clinical use of ERT may require ApoE genotyping for optimal efficacy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EPIDEMIOLOGICAL STUDIES SUGGEST that estrogen replacement therapy (ERT) decreases the risk for or the rate of progression in several chronic neurological diseases. The majority of epidemiological studies suggest a history of ERT decreases the likelihood of developing late-life dementia (1, 2, 3, 4, 5, 6). However, in the Women’s Health Initiative trial, the continuous estrogen-plus-progestin arm, which was discontinued early, found no protection from dementia and perhaps a slight increase in risk (7, 8). A history of ERT also may delay either the onset or progression of Parkinson disease (PD) (9, 10, 11, 12). These studies are also mixed with some reporting no change in risk (9). Finally, the rate of clinical progression of multiple sclerosis is attenuated by estrogen (13, 14). The disagreement in studies emphasizes our lack of a clear understanding of the mechanism whereby steroid replacement may decrease risk of neurological diseases.

Apolipoprotein E (ApoE) modifies the risks for several chronic neurological diseases. Humans have three major isoforms of ApoE (ApoE2, ApoE3, and ApoE4) that are produced by three alleles, {epsilon}2, {epsilon}3, and {epsilon}4, at a single gene locus on chromosome 19 (15, 16). Inheritance of {epsilon}4 increases the risk and rate of progression of late-onset Alzheimer disease (17, 18, 19). Individuals with {epsilon}4 alleles also appear to have an earlier age of onset of PD and a greater risk of developing dementia in PD (20, 21). Additionally, {epsilon}4 inheritance increases the risk of developing dementia after head trauma (22, 23, 24) and increases the severity of relapsing-remitting multiple sclerosis (25, 26).

Neuronal repair may be a common factor in these diseases. Estradiol and ApoE independently facilitate neurite/process outgrowth in vivo and in vitro. Estradiol increases the complexity of neuritic growth and axonal elongation in vitro (27, 28, 29). Neuronal repair after brain lesion is more rapid in the presence of estradiol (30, 31). ApoE, similar to estrogen, has dramatic effects on neurite outgrowth from a variety of cultured neurons. ApoE3 increases neurite outgrowth, whereas the absence of ApoE or treatment with ApoE4 either decreases or has no effect on neurite outgrowth (32, 33, 34). Neuropathological studies have documented deficits in dendritic growth and plasticity in brains of ApoE4 individuals (35). ApoE appears to be a critical element in neuronal growth and repair.

ApoE levels in the CNS vary during the estrous cycle (36) and several studies show that estradiol can increase ApoE in vivo (36, 37) and in vitro (38, 39). In sum, these observations suggest a generalized mechanism whereby estrogen could increase ApoE levels. ApoE levels could then facilitate process regrowth after injury. We tested this hypothesis in cultured neurons from adult mice. We found that the presence of ApoE2 or ApoE3 was necessary to obtain an effect of estrogen on neurite outgrowth. In addition, our data suggest that ApoE isoforms have differential rates of internalizaton and lipid transport into neurons.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects
An in vitro culture paradigm of adult cortical neurons (40) was used to examine the effect of 17ß-estradiol (estradiol) on ApoE levels and neurite extension. The adult cortical culture procedure has been previously described (33, 40). All procedures were approved by the Eastern Illinois University Animal Care Committee.

Control mice (C57BL/6) and homozygous ApoE knockout (KO) mice, bred 10 generations onto the C57BL/6 background (C57BL/6-Apoe<tmiUnc>) were obtained from The Jackson Laboratory (Bar Harbor, ME). Transgenic (tg) human ApoE3 and tg human ApoE4 mice were a gift from Dr. David Holtzman at Washington University (St. Louis, MO). The ApoE3 or ApoE4 transgene was inserted with a glial fibrillary acidic protein (GFAP) promoter region (34).

Culture preparation
For each experiment, a 4-month-old female mouse was euthanized with sodium pentobarbital (80 mg/kg). The entire cerebral cortex was dissected from the brain and placed in 2 ml B27/Hibernate A medium (B27/HA, Invitrogen Life Technologies, Carlsbad, CA) with 0.5 mM glutamine (Sigma, St. Louis, MO) at 4 C. The cortex was sliced (0.5 mm thickness) and transferred to a 50-ml tube containing 5 ml B27/HA. After warming for 8 min at 30 C, slices were digested with 6 ml of a 2 mg/ml papain (Sigma) solution in B27/HA for 30 min at 30 C in a gyrating water bath. The slices were transferred to 2 ml B27/HA. After 2 min at room temperature, the slices were triturated 10 times with a siliconized 9-inch Pasteur pipette, and allowed to settle for 1 min. Approximately 2 ml of the supernatant were transferred to another tube, and the sediment resuspended in 2 ml B27/HA. The above step was repeated twice, and a total of 6 ml collected. The resultant supernatant was subjected to density gradient centrifugation at 800 x g for 15 min. The density gradient was prepared in four 1-ml layers of 35, 25, 20, and 15% Optiprep (Invitrogen Life Technologies) in B27/HA medium (vol/vol). Debris above 7 ml was discarded. The rest of the fractions, excluding the bottom pellet, were collected and diluted in 5 ml of B27/HA. After centrifuging twice at 200 x g for 2 min, the cell pellets were resuspended in 3 ml B27/neurobasal A medium (Invitrogen Life Technologies) with 0.5 mM glutamine and 0.01 mg/ml gentamicin (Sigma). A total of 4 x 104 cells were plated in 50-µl aliquots on glass cover slips (12 mm diameter) that were coated overnight with poly-D-lysine (50 mg/ml, Sigma). After 1 h incubation in a humidified incubator at 37 C and 5% CO2, each cover slip was rinsed twice with B27/HA then transferred to a 24-well plate containing 0.4 ml B27/neurobasal A medium with 5 ng/ml fibroblast growth factor 2 (Invitrogen Life Technologies).

Estradiol-17ß (Sigma), dissolved in 95% ethanol at 40 µM, was added to the culture medium after 1 d in vitro (1 DIV), and the incubation was continued until 4 DIV. Human recombinant ApoE3 and ApoE4 (Panvera, Madison, WI) were dialyzed overnight in 0.1 M ammonium bicarbonate. The final concentrations of estrogen, ApoE, and other test reagents in the culture medium varied based on experiments. All quantification was performed on cells at 4 DIV. Each experiment was performed three times.

ApoE quantification
Media from 4 DIV cultures were collected and centrifuged for 5 min. ApoE was immunoprecipitated by incubating the medium with monoclonal anti-ApoE (3H1, Heart Institute, University of Ottawa, Ottawa, Canada) at 1:1000 dilution in the media on ice for 1 h. After incubation, 10% Protein A-Sepharose CL-4B (Sigma) was added (1:100), and the medium was further incubated on ice for 1 h on a shaker. The medium was centrifuged at 11,000 x g for 15 min at 4 C and the supernatant discarded. The pellet was boiled for 5 min in 2x Lammeli sample buffer and electrophoresed on a 4–20% sodium dodecyl sulfate-polyacrylamide gradient gel in an EC120 Mini gel vertical system. The samples were run at 80 V until separation began, and then at 140 V until the dye front neared the bottom of the gel.

After electrophoresis, the gel was placed in transfer buffer (3.03 g Tris-base, 14.4 g glycine, 200 ml methanol, 800 ml distilled H2O) on a shaker for 10 min. The transfer membrane, Immobilon-P Transfer Membrane (Millipore, Bedford, MA), was first soaked in methanol for 5 sec then washed in distilled H2O for 5 min to remove excess methanol. The proteins in the gel were transferred to the membrane using a Bio-Rad (Hercules, CA) Trans-blot Transfer Cell following the manufacturer’s protocol. The membranes were blocked in TBS buffer [20 mM Tris-HCl, 150 mM NaCl (pH 7.5)] with 1% BSA at 4 C overnight or at room temperature for 1 h. After blocking, the membranes were washed twice (5 min each) with T-TBS buffer (TBS buffer with 0.05% Tween 20) and incubated in goat antihuman ApoE (Calbiochem, San Diego, CA) at 1:5000 dilution in T-TBS buffer for 1 h on a shaker at room temperature. The membranes were washed four times (10 min each) in T-TBS buffer and incubated in horseradish peroxidase-conjugated rabbit antigoat IgG (Chemicon, Temecula, CA) at 1:10,000 dilution in T-TBS buffer for 1 h on a shaker at room temperature. After washing five times (10 min each) with T-TBS buffer, the membranes were incubated with SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL), and exposed to Kodak BioMax Light-2 film (Fisher Scientific, Chicago, IL). A 34-kDa band was visualized and is consistent with the published molecular mass of ApoE. Bands were quantified by densitometry (Scion Image, Frederick, MD) and compared with a band of recombinant human ApoE.

Neurite extension
Cells were prepared as described above and treated with varying concentrations of estradiol. Ethanol (0.1 nM), the vehicle for estradiol was included as an additional control. Single neurite extension (the length of the longest neurite of a neuron) and combined neurite length (the total length of all neurites from a neuron) were measured as described previously (33). The first technique employed an ocular micrometer to measure the longest neurite. The second technique measured the total length of all neurites. A minimum of ten cells per coverslip were quantified. The values from each coverslip were averaged to yield the mean longest length and mean total length. Each experiment was performed three times. The following experiments were performed with this culture system.

Experimental studies
Time course of ApoE levels after estradiol treatment.
The time course of ApoE levels in response to exogenous estradiol was examined. ApoE secretion in adult mouse cultures was evaluated by incubating the cells with 0.1 nM estradiol for 0, 0.1, 0.5, 1.0, 4.0, 16.0, 36.0, and 48.0 h. After incubation, the medium was collected, and secreted ApoE was quantified by immunoblotting after immunoprecipitation.

Dose-response of ApoE to estradiol treatment.
We obtained a dose-response curve of ApoE levels to estradiol addition from wild-type mice after 3 d of estradiol treatment at dosages of estradiol ranging from 0.1 pM to 10 nM. Cortical cells from wild-type mice were treated with varying concentrations of estradiol (0.1 pM to 10 nM). After 3 d, the amount of secreted ApoE was measured as described above.

Dose-response of estradiol on neurite extension.
A second dose-response study of estradiol was performed on neurite outgrowth at 4 DIV. Cultures were prepared and varying concentrations of estradiol were added to the cells at 1 DIV. At 4 DIV, cells were evaluated for neurite extension as described above.

ApoE replacement on neurite extension.
We performed ApoE replacement studies in estradiol-treated cell cultures derived from mice lacking the ApoE gene (ApoE KO; The Jackson Laboratory). Cells were incubated with or without 100 pM of estradiol in ethanol (starting at 1 DIV). Purified recombinant human ApoE isoforms were added at 3 µg/ml (Panvera). This concentration is comparable to that found in human cerebrospinal fluid (41). After 4 DIV, the combined length of neuritic trees was obtained as described above.

Estradiol effects on neurite outgrowth in tg mice.
tg Mice expressing human ApoE3 or ApoE4, under the control of glial fibrillary acidic protein promoter (34) were used to evaluate the effects of endogenous ApoE on estradiol-stimulated neurite extension in culture. In vivo, these mice show ApoE protein expression in astrocytes (Ref. 34 ; and Struble, R. G., and B. P. Nathan, personal observations). Cultures were prepared for evaluation at 4 DIV as above.

ApoE/lipid uptake studies.
Because ApoE transfers lipids to neurons by endocytosis, we tested the rate of uptake of exogenous ApoE and lipid by neurons in cultures derived from ApoE KO mice. We examined both the rate of ApoE uptake and the rate at which a fatty acid was transported into cultured neurons. Cultures at 4 DIV from ApoE KO mice were incubated for 1 h with recombinant human ApoE3 or human ApoE4, and then prepared for immunocytochemical localization of ApoE. Coverslips were fixed with 80% ethanol for 1 h at RT, rinsed three times in T-TBS [0.2% Tween in 20 mM TBS (pH 7.5)] then permeabilized with 0.25% Triton in PBS (0.1 M, pH 7.2) for 30 min. Coverslips were then rinsed three times in T-TBS. Nonspecific binding was blocked with 1% BSA in 15% ApoE KO mouse serum in T-TBS for 1 h at RT and then incubated for 2 h at room temperature with a goat anti-ApoE antibody (Chemicon) at 1:200 dilution. After washing six times with T-TBS the coverslips were incubated with fluorescently labeled antigoat antisera (Pierce) for 30 min at 1:200 dilution and then washed five times in T-TBS for 5 min each. Control coverslips were incubated at 4 C. Coverslips were mounted with anti-photobleach medium (Vectashield, Vector Laboratories, Burlingame, CA) and stored in the dark. Digital images of 30 fields per coverslip (x40 objective) containing neurons were obtained using a fluorescence microscope with an excitation frequency of 520 nm and emission at 492 nm with a digital camera (Pixera, Penguin 150CL) maintaining identical exposure settings for all images. ApoE immunofluorescence was measured by quantifying mean optical fluorescence of each field using Scion Image software. The upper and lower 20 bits were cut off to eliminate nonspecific fluorescence and a mean density of pixels/field within this range were obtained. The data were transformed to a scale of 0 to 100 for statistical analysis.

Lipid uptake by neurons was quantified with fluorescently labeled dodecanoic acid (4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid) accumulation in adult cortical neurons. Fluorescence absorption and emission were at 500 and 510 nM, respectively. Cells grown on coverslips for 4 d were incubated for 1 h with 3 µg/ml of either ApoE 3 or ApoE 4 together with 10 µg/ml of a fluorescently labeled lipid, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (Molecular Probes, Eugene, OR). After incubation, the coverslips were rinsed three times with warm 37 C PBS, and the fluorescent images were digitized with an objective magnification of x100 as described above. OD was measured using Scion Image software as described above.

Receptor inhibition on ApoE uptake.
To determine the type of lipoprotein receptors underlying the effects of ApoE isoforms on neuronal internalization of lipoproteins, three different lipoprotein receptor inhibitors were used: 1) receptor-associated protein (RAP, 5 µg/ml; a gift from Dr. Dudley Strickland, American Red Cross, Rockville, MD), 2) lactoferrin (LAC, 10 µg/ml, Sigma) and 3) heparinase I (10 U/ml, Sigma). Heparinase depletes cell-surface heparan sulfate proteoglycans (HSPGs) that bind ApoE-enriched lipoproteins before internalization via the LRP, whereas RAP and LAC inhibits the binding of lipoproteins to the LRP. Cells (3 DIV) from ApoE KO mice were preincubated for 1 h in medium alone or in medium containing one of the three inhibitors. Cultures were subsequently treated for 1 h with recombinant ApoE3 or ApoE4, and prepared for immunofluorescent localization of ApoE. Images of 10 neurons per coverslip were digitized and analyzed for each experimental condition.

Statistical analysis
Data from each experiment were analyzed by ANOVA. Per cell, n = 3 in all cases, representing the three replications of each experiment. When appropriate, as noted by a significant effect found with ANOVA, pair-wise post hoc testing was performed with Bonferroni-corrected t tests to maintain an overall type I error rate of P < 0.05. In the presence of a significant interaction, the data were decomposed and treated as a one-way analysis with Bonferroni-corrected t tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time course of ApoE levels to estradiol treatment
This experiment showed that ApoE was rapidly produced after the introduction of estradiol. ANOVA with time as the independent factor identified a significant effect (F7,16 = 81.939; P < 0.001). Post hoc t testing disclosed that 4 h or more of estradiol exposure resulted in a significant increase in ApoE levels compared with earlier time periods (Fig. 1Go). ApoE levels between 4 and 48 h did not differ. Western blots of ApoE showed the expected band at 34 kDa (Fig. 2Go).



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  		FIG. 1. Time course of ApoE levels in cultures in response to 100 pM of 17ß-estradiol added at one DIV and sampled between 6 min and 96 h. ApoE levels significantly increased 4 h after addition of estradiol and remain elevated. Data are presented as mean ± SEM to show the variability at each point. Before 4 h, variability among cultures in ApoE levels is extremely small.

 


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  		FIG. 2. Western blotting of ApoE from mixed cell cultures after treatment with estradiol. Little or no ApoE is produced before a 4-h exposure to estradiol.

 
Dose-response of ApoE levels to estradiol treatment
We next examined the effects of increasing concentrations of estradiol on ApoE levels in the culture (Fig. 3AGo). ANOVA found an overall significance of estradiol concentration on ApoE levels (F5,12 = 260.546; P < 0.001). Post hoc testing with a Bonferroni corrected t test showed each higher dose of estradiol significantly increased ApoE levels from the lower dose except that 100 pM was not different from 1 pM of estradiol. A dose of 1 nM of estrogen exhibited the highest response of ApoE levels. A 10-nM concentration of estrogen had a significantly smaller effect on ApoE levels than 1 pM–1 nM of estradiol. Normal female mouse estradiol concentrations are between 10 and 80 pM throughout the estrous cycle (42). Hence, these data suggest that a physiological concentration of estradiol had the greatest effect, and that a supraphysiologic concentration was less effective.



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  		FIG. 3. A, Increasing concentrations of estradiol significantly increase ApoE levels in culture. Post hoc testing with a Bonferroni corrected t test showed each higher dose was significant (*) from the lower dose except that 100 pM was not different from 1 pM. Response to 1 nM of estrogen (**) was significantly higher than all other doses. A 10-nM concentration of estrogen had a significantly smaller effect (***) on ApoE levels than1 pM-1 nM of estradiol. B, Estradiol stimulates combined neuritic length. One-way ANOVA showed significance at P < 0.001. Post hoc testing by Bonferroni corrected t tests showed that 100 pM and 1 nM of estrogen significantly (*) increased neuritic outgrowth compared with the control conditions of no estradiol or ethanol alone. Note that peak responses to estrogen in both ApoE production and neuritic outgrowth occurred at identical estrogen concentrations.

 
Dose-response of estradiol on neurite extension
This experiment examined the effect of increasing doses of estradiol on neurite outgrowth (Fig. 3BGo). Neurons were easily identifiable in culture by differential interference contrast microscopy (Fig. 4Go) and have been previously confirmed with immunocytochemistry (33). Longest neurite and combined length correlated highly (r = 0.98); only combined length is reported. One-way ANOVA showed significance of estrogen treatment on neurite extension (F6,12 = 8.012; P < 0.001). Post hoc testing indicated that 100 pM and 1 nM of estrogen significantly increased neuritic outgrowth compared with the control conditions of no estradiol or ethanol alone, whereas the response to 10 nM of estrogen did not differ from that of the control conditions. Therefore, neuritic extension showed the same peak dosage effect of estradiol as did ApoE levels.



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  		FIG. 4. Differential interference contrast digital image of two adjacent neurons in culture at 4 DIV. This is the typical appearance of neurons in adult mouse cultures. A previous study (33 ) used antineurofilament antibody to confirm this conclusion. Note the microglia at the lower left of the figure.

 
ApoE replacement on neurite extension
Previous studies have shown that ApoE isoforms have differential effects on neurite outgrowth (32, 33, 34, 43). This experiment determined the combined effects of 100 pM estradiol and ApoE on neurite growth in cultures from ApoE KO mice (Fig. 5Go). ANOVA showed a significant main effect of estradiol on neuritic length (F1,16 = 125.344; P < 0.001), of ApoE isoform (F = 256, F3,16; P < 0.001) and the interaction between estradiol treatment and ApoE isoform (F3,16 = 45.315; P < 0.001). Decomposition of the interaction term by post hoc one-way ANOVA and post hoc t tests showed that ApoE2 or ApoE3 alone increased combined neurite length by about 30% compared with cultures without ApoE or with ApoE4 (F3,8 = 27.649, P < 0.001). ApoE2 did not differ from ApoE3 on post hoc t testing. These data replicate previous results (32, 33). Analysis of the estradiol-only group also showed an isoform-specific effect (F3,8 = 700.702, P < 0.001). Bonferroni corrected post hoc t testing showed that combined neurite length in the KO group was equivalent to the ApoE4 replacement group and was less than the ApoE2 (P < 0.001) or ApoE3 (P < 0.002) replacement group. Estradiol increased neuritic length when combined with ApoE2 (+95%) or ApoE3 (+79%) but not in the absence of ApoE or treatment with ApoE4. Post hoc t testing found the difference between ApoE2 and ApoE3 significant (P < 0.003). In sum, estradiol increased neurite outgrowth, but only in the presence of ApoE2 or ApoE3 and estradiol had a greater effect on neurite growth in the presence of ApoE2 when compared with ApoE3.



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  		FIG. 5. Estradiol has an ApoE isoform-specific effect on neuritic outgrowth in cultures derived from ApoE KO mice. ANOVA showed an overall significant interaction of ApoE replacement and estradiol treatment (P < 0.001). Post hoc t testing found that estradiol had no effect on cultures lacking ApoE or supplemented with ApoE4. Inclusion of ApoE2 or ApoE3 in the absence of estrogen significantly increased neuritic extent (*) compared with no ApoE or ApoE4. Estradiol increased neurite outgrowth beyond that of ApoE alone in the ApoE2 or ApoE3 conditions (**). In addition, the neuritic extent was significantly greater (P < 0.003) in the ApoE2 than the ApoE3 group (line).

 
Estradiol effects on neurite outgrowth in tg mice
To determine whether endogenously produced ApoE isoforms can differentially modulate estradiol-induced neurite outgrowth we added estradiol to neuronal cultures derived from tg mice expressing either ApoE3 or ApoE4. After treatment of neuronal cultures with estradiol, secreted ApoE levels (Fig. 6AGo) were comparable in both genotypes (F1,8 = 0.162;. P = 0.698). Estradiol (100 pM) significantly increased ApoE levels by about 30% in cultures from both genotypes (F1,8 = 81.619; P < 0.001). There was no interaction between estradiol treatment and ApoE levels for the two genotypes (F1,8 = 0.030; P = 0.867).



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  		FIG. 6. A, Amounts of ApoE produced by estradiol stimulation in ApoE-tg mice. Treatment with 100 pM of 17ß-estradiol increased ApoE levels in the ApoE-tg mice cultures by about 30% compared with vehicle-treated cultures. The effect is not genotype specific. B, Neuritic extent in ApoE-tg mice. ApoE3-tg mice cultures had a greater combined neurite outgrowth than ApoE4-tg mice (*). Estradiol increased this difference (**). Estradiol had no effect on neuritic extent in the ApoE4-tg cultures although it increased ApoE levels equal to that of the estradiol-treated ApoE3-tg cultures.

 
Combined length of neurites (Fig. 6BGo) was significantly greater in the ApoE3-tg than in the ApoE4-tg mouse cultures (F1,8 = 390.543; P < 0.001). Estradiol treatment increased total length (F1,8 = 58.292; P < 0.001). There was a significant interaction between genotype and estradiol treatment. There was no effect on combined neuritic length in ApoE4-tg mouse cultures, whereas neuritic extent was increased in the ApoE3-tg mouse cultures (F1,8 = 53.937; P < 0.001). In essence, neurites were longer in the cultures from ApoE3-tg mice and estradiol increased that differential only in the cultures from ApoE3-tg mice.

ApoE/lipid uptake studies
In this experiment, we evaluated the rate of internalization of two ApoE isoforms, ApoE3 and ApoE4 in the adult culture model to replicate previous results with higher concentrations of ApoE in neuroblastoma cells (32). We also evaluated the rate of uptake of a fluorescently labeled fatty acid as a function of ApoE isoform. There was less intracellular accumulation of ApoE4 than ApoE3 (Fig. 7AGo). ApoE-like immunoreactivity within neurons incubated with ApoE3 was significantly greater (F1,20 = 6.420; P < 0.02) than ApoE4 as previously reported (32). ANOVA showed neither a significant effect of time post addition of ApoE (F4,20 = 2.032; P = 0.128) nor an interaction between time and type of ApoE (F4,20 = 0.697; P = 0.603). ApoE-like immunoreactivity was intense in the cell body, and extended into the processes in vesicular-like accumulations (Fig. 8Go).



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  		FIG. 7. A, Accumulation of ApoE isoforms by neurons. ANOVA showed a significant effect of ApoE type (P < 0.02). More ApoE3 immunoreactivity was found within neurons than ApoE4. No other factor reached statistical significance. B, Lipid accumulation at 1 h after incubation with ApoE isoforms or in the absence of ApoE (KO). Recombinant ApoE3 facilitated fluorescently labeled dodecanoic acid accumulation by neurons when compared with the recombinant ApoE4. Post hoc t testing showed ApoE3>ApoE4>ApoE KO for total lipid uptake.

 


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  		FIG. 8. Immunofluoresence localization of ApoE3 or ApoE4 in neurons. Neuronal internalization of ApoE4 is less efficient than ApoE3. At time zero (A and B) the intensity of intracellular immunoreactivity of both ApoE3 and ApoE4 was roughly comparable. Greater uptake of ApoE 3 than E4 was seen at 1 h (C and D). The ApoE is also present in vesicular-like structures in the neurites. Bar in D, 20 µm.

 
A comparable ApoE isoform-specific effect for fatty acid accumulation in neurons was present (F2,6=142.173; P < 0.001). Accumulation of fluorescent lipid in cells cultured with ApoE3 was approximately twice (P < 0.001; post hoc t test) that in cells treated with ApoE4, and displayed the same vesicular appearance in the processes of the neurons as did ApoE (Fig. 9Go). ApoE4 roughly doubled (P < 0.001, post hoc t test) the amount of lipid internalized compared with the absence of ApoE (Fig. 7BGo). ApoE4 was much less effective than ApoE3 in intracellular transport of lipids, although it was more effective than no ApoE treatment.



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  		FIG. 9. Uptake of fluorescently labeled dodecanoic acid. Uptake in the absence of ApoE (A) is less than when ApoE3 (B) or ApoE4 (C) is present. The perikaryal size is equivalent for each condition but appears greater for ApoE3 because of more fluorescent lipid in the cell body. Note that the fluorescence is vesicular like in the neurites. Bar in C, 20 µm.

 
Receptor inhibition on ApoE uptake
This experiment was performed to determine the receptor specificity of ApoE uptake in adult neuronal cultures. Neither ApoE3- nor ApoE4-like immunoreactivity was found in neurons treated with heparinase I; therefore, no statistical analysis was performed. A repeated measures ANOVA of ApoE accumulation in neurons for RAP or LAC compared with no inhibitor (with inhibition condition as the repeated measure) showed a significant main effect of ApoE isoform (F1,4; = 383.123; P < 0.001) (Fig. 10AGo). Overall, less ApoE4 entered neurons than ApoE3. A significant effect of inhibitor compared with no inhibitor was found (F2,8 = 2381.714; P < 0.001). In addition, there was a significant interaction between isoform and inhibitor (F2,4 = 372.474; P < 0.001). Calculating percent inhibition (Fig. 10BGo) from the untreated condition showed that LAC inhibited ApoE3 uptake by 91% and ApoE4 uptake by 97%. In contrast, RAP inhibited ApoE3 uptake by only 76% but ApoE4 by 99%.



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  		FIG. 10. A, Inhibition of ApoE immunoreactivity in cells by LAC and RAP. Note that RAP has a greater effect on ApoE4 than on ApoE3. Roughly equivalent inhibitory effects on both ApoE isoforms were shown by LAC. B, Plotting % inhibition in B emphasizes the differential effect of RAP on ApoE3 vs. ApoE4.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Numerous studies have shown that estradiol modifies neuronal growth in culture (see Ref. 44). Generally, these effects increase survival or process extension. The general assumption of these studies is that there is only a direct effect of estradiol on neuronal activation and growth. This assumption may be too simplistic. Our data suggest that ApoE plays an important role in neuronal activation and growth stimulated by estradiol.

Previous data suggest that glia are the primary source of ApoE (45, 46, 47) and our studies with ApoE tg mice support this. Therefore, we selected a mixed culture derived from adult mouse brain to evaluate estradiol effects on ApoE levels and neuronal process growth. Adult cortical neuronal cultures contain both microglia and astroglia early in the culture. Our previous study showed that neurons comprise 70% of total cells, astroglia 15% and microglia 15% at 4 DIV (33). Glial-derived factors are important in synaptogenesis (see Ref. 48) and inclusion of glia in the culture may be required to see a broader spectrum of the effects of estrogen on neurons in vitro. For example, a previous report has shown that both microglia and astroglia must be present to obtain an estrogen-induced increase in ApoE mRNA (38). Hence, a monotypic cell culture may give a biased interpretation of critical estradiol effects if interactions exist between glia and neurons.

Data from previous studies suggest a direct relationship between ApoE and neurite outgrowth (32, 33, 34, 49, 50). We found that a physiological concentration of estradiol had a maximal effect on both ApoE levels and neurite outgrowth. Our data also suggest a synergistic effect between estradiol and ApoE such that estradiol effects on neurite outgrowth are facilitated by ApoE3 but not by ApoE4. ApoE3 alone increased neurite extent by about 30%. But when combined with estradiol, neuritic growth increased by 70–80%. In addition, ApoE2 was significantly more effective than ApoE3 in facilitating neurite outgrowth in the presence of estradiol. This effect corresponds to studies that suggest a protective effect of ApoE2 in dementia (see Ref. 51). In addition, the differential effects of ApoE3 vs. ApoE4 were confirmed by using tg mice expressing ApoE3 or ApoE4. Estrogen increased the levels of both ApoE3 and ApoE4 to a similar extent in adult cultures derived from ApoE3- and ApoE4-tg mice; however, only the ApoE3 isoform was effective in facilitating neurite outgrowth in the presence of estradiol.

A partial mechanism for differential effects of ApoE3 and ApoE4 is suggested by our uptake studies. The procedure used for preparing adult cultures strips cell processes. During the next 4 d, extensive neurite sprouting occurs. Estradiol may stimulate neurons to extend processes and concurrently stimulate astroglia (and perhaps microglia) to produce ApoE. Glial-derived ApoE supplies lipids for neuronal membrane synthesis and function. Our receptor inhibition studies suggest that both ApoE3 and ApoE4 are using low-density lipoprotein receptor-related protein and HSPG for neurite outgrowth (33, 49). However, neuronal growth is slowed or disrupted in the absence of ApoE or in the presence of ApoE4. It is worth noting that ApoE4 has been reported to show toxic effects in culture (52) and disrupt microtubule polymerization although at 10-fold higher concentrations of those used in this study (32). Hence, the less complex neuritic field seen in the cells incubated with ApoE4 may represent a deficiency of lipid in combination with toxic effects of ApoE4.

Process extension during neuronal repair in vivo may be a critical factor for the rate of progression in some chronic neurological diseases. In dementia or after trauma, degeneration and repair may be in dynamic balance. Dementia or cognitive decline may occur when degenerative processes exceed regenerative responses. ApoE4 may tip the scales to slower or incomplete regeneration and accelerate cognitive decline more rapidly than ApoE2 or ApoE3. Of interest is that ApoE4 has been shown to be a dominant-negative factor in mice expressing both ApoE3 and ApoE4 (49, 53). ERT may be less protective in those with {epsilon}4 allele, which has been previously noted (54). The full benefit of ERT in protection from some chronic neurological diseases may be compromised by the presence of ApoE4. Clinical studies to evaluate protective effects of ERT or estradiol-like compounds may need to consider ApoE genotype to obtain optimal efficacy.


   Acknowledgments
 
The GFAP-ApoE3 and GFAP-ApoE4 tg mice were generously provided by Dr. David M. Holtzman (Washington University, St. Louis, MO). RAP was a generous gift from Dr. Dudley Strickland (American Red Cross, Rockville, MD).


   Footnotes
 
This research was funded by the Illinois Department of Public Health Alzheimer’s Fund (to B.P.N. and R.G.S.), National Institutes of Health Academic Research Enhancement Award (to B.P.N.), Eastern Illinois University council of faculty research grants (to B.P.N.), and the Southern Illinois University School of Medicine, Central Research Committee (to R.G.S.).

Abbreviations: apoE, Apolipoprotein E; DIV, days in vitro; ERT, estrogen replacement therapy; GFAP, glial fibrillary acidic protein; HA, Hibernate A; HSPG, heparan sulfate proteoglycan; KO, knockout; LAC, lactoferrin; PD, Parkinson disease; RAP, receptor-associated protein; tg, transgenic.

Received December 17, 2003.

Accepted for publication March 11, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Paganini-Hill A, Henderson V 1994 Estrogen deficiency and risk of Alzheimer’s disease in women. Am J Epidemiol 140:256–261[Abstract/Free Full Text]
  2. Brenner DE, Kukull WA, Stergachis A, van Belle G, Bowen JD, McCormick WC, Teri L, Larson EB 1994 Postmenopausal estrogen replacement therapy and the risk of Alzheimer’s disease: a population-based case-control study. Am J Epidemiol 140:262–267[Abstract/Free Full Text]
  3. Tang MX, Jacobs D, Stern Y, Marder K, Schofield P, Gurland B, Andrews H, Mayeux R 1996 Effect of oestrogen during menopause on risk and age at onset of Alzheimer’s disease. Lancet 348:429–432[CrossRef][Medline]
  4. Kawas C, Resnick S, Morrison A, Brookmeyer R, Corrada M, Zonderman A, Bacal C, Lingle DD, Metter E 1997 A prospective study of estrogen replacement therapy and the risk of developing Alzheimer’s disease: the Baltimore Longitudinal Study of Aging. Neurology 48:1517–1521[Abstract]
  5. LeBlanc ES, Janowsky J, Chan BK, Nelson HD 2001 Hormone replacement therapy and cognition: systematic review and meta-analysis. JAMA 285:1489–1499[Abstract/Free Full Text]
  6. Zandi PP, Carlson MC, Plassman BL, Welsh-Bohmer KA, Mayer LS, Steffens DC, Breitner JC 2002 Hormone replacement therapy and incidence of Alzheimer disease in older women: the Cache County Study. JAMA 288:2123–2129[Abstract/Free Full Text]
  7. Rapp SR, Espeland MA, Shumaker SA, Henderson VW, Brunner RL, Manson JE, Gass ML, Stefanick ML, Lane DS, Hays J, Johnson KC, Coker LH, Dailey M, Bowen D, WHIMS Investigators 2003 Effect of estrogen plus progestin on global cognitive function in postmenopausal women: the Women’s Health Initiative Memory Study: a randomized controlled trial. JAMA 289:2663–2672[Abstract/Free Full Text]
  8. Shumaker SA, Legault C, Rapp SR, Thal L, Wallace RB, Ockene JK, Hendrix SL, Jones 3rd BN, Assaf AR, Jackson RD, Kotchen JM, Wassertheil-Smoller S, Wactawski-Wende J, WHIMS Investigators 2003 Estrogen plus progestin and the incidence of dementia and mild cognitive impairment postmenopausal women: the Women’s Health Initiative Memory Study: a randomized controlled trial. JAMA 289:2651–2662[Abstract/Free Full Text]
  9. Mayeux R, Marder K, Cote LJ, Denaro J, Hemenegildo N, Mejia H, Tang MX, Lantigua R, Wilder D, Gurland B 1995 The frequency of idiopathic Parkinson’s disease by age, ethnic group, and sex in Northern Manhattan, 1988–1993. Am J Epidemiol 142:820–827[Abstract/Free Full Text]
  10. Kuopio AM, Marttila RJ, Helenius H, Rinne UK 1999 Changing epidemiology of Parkinson’s disease in southwestern Finland. Neurology 52:302–308[Abstract/Free Full Text]
  11. Saunders-Pullman R, Gordon-Elliott J, Parides M, Fahn S, Saunders HR, Bressman S 1999 The effect of estrogen replacement on early Parkinson’s disease. Neurology 52:1417–1421[Abstract/Free Full Text]
  12. Marder K, Tang MX, Alfaro B, Mejia H, Cote L, Jacobs D, Stern Y, Sano M, Mayeux R 1998 Postmenopausal estrogen use and Parkinson’s disease with and without dementia. Neurology 50:1141–1143[Abstract/Free Full Text]
  13. Smith R, Studd JW 1992 A pilot study of the effect upon multiple sclerosis of the menopause, hormone replacement therapy and the menstrual cycle. J R Soc Med 85:612–613[Abstract]
  14. Sicotte NL, Liva SM, Klutch R, Pfeiffer P, Bouvier S, Odesa S, Wu TC, Voskuhl RR 2002 Treatment of multiple sclerosis with the pregnancy hormone estriol. Ann Neurol 52:421–428[CrossRef][Medline]
  15. Mahley RW 1988 Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science 240:622–630[Abstract/Free Full Text]
  16. Weisgraber KH 1994 Apolipoprotein E: structure-function relationships. Adv Protein Chem 45:249–302[Medline]
  17. Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE, Gaskell PC, Small GW, Roses AD, Haines JL, Pericak-Vance MA 1993 Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families. Science 261:921–923[Abstract/Free Full Text]
  18. Roses AD 1997 Apolipoprotein E, a gene with complex biological interactions in the aging brain. Neurobiol Dis 4:170–185[CrossRef][Medline]
  19. Strittmatter WJ, Roses AD 1996 Apolipoprotein E and Alzheimer’s. Annu Rev Neurosci 19:53–77[CrossRef][Medline]
  20. Tang G, Xie H, Xu L, Hao Y, Lin D, Ren D 2002 Genetic study of apolipoprotein E gene, {alpha}-1 antichymotrypsin gene in sporadic Parkinson disease. Am J Med Genet 114:446–449[CrossRef][Medline]
  21. Zareparsi S, Camicioli R, Sexton G, Bird T, Swanson P, Kaye J, Nutt J, Payami H 2002 Age at onset of Parkinson disease and apolipoprotein E genotypes. Am J Med Genet 107:156–161[CrossRef][Medline]
  22. Mayeux R, Ottman R, Maestre G, Ngai C, Tang MX, Ginsberg H, Chun M, Tycko B, Shelanski M 1995 Synergistic effects of traumatic head injury and apolipoprotein-{epsilon}4 in patients with Alzheimer’s disease. Neurology 45:555–557[Abstract/Free Full Text]
  23. Teasdale GM, Nicoll JA, Murray G, Fiddes M 1997 Association of apolipoprotein E polymorphism with outcome after head injury. Lancet 350:1069–1071[CrossRef][Medline]
  24. Jordan BD, Relkin NR, Ravdin LD, Jacobs AR, Bennett A, Gandy S 1997 Apolipoprotein E {epsilon}4 associated with chronic traumatic brain injury. JAMA 278:136–140[Abstract]
  25. Fazekas F, Strasser-Fuchs S, Kollegger H, Berger T, Kristoferitsch W, Schmidt H, Enzinger C, Schiefermeier M, Schwarz C, Kornek B, Reindl M, Huber K, Grass R, Wimmer G, Vass K, Pfeiffer KH, Hartung HP, Schmidt R 2001 Apolipoprotein E epsilon 4 is associated with rapid progression of multiple sclerosis. Neurology 57:853–857[Abstract/Free Full Text]
  26. Masterman T, Zhang Z, Hellgren D, Salter H, Anvret M, Lilius L, Lannfelt L, Hillert J 2002 APOE genotypes and disease severity in multiple sclerosis. Mult Scler 8:98–103[Abstract/Free Full Text]
  27. Ferreira A, Caceres A 1991 Estrogen-enhanced neurite growth: evidence for a selective induction of {tau} and stable microtubules. J Neurosci 11:392–401[Abstract]
  28. Diaz H, Lorenzo A, Carrer HF, Caceres A 1992 Time lapse study of neurite growth in hypothalamic dissociated neurons in culture: sex differences and estrogen. J Neurosci Res 33:266–281[CrossRef][Medline]
  29. Brinton RD, Tran J, Proffitt P, Montoya M 1997 17beta]-Estradiol enhances the outgrowth and survival of neocortical neurons in culture. Neurochem Res 22:1339–1351[CrossRef][Medline]
  30. Morse JK, DeKosky ST, Scheff SW 1992 Neurotrophic effects of steroids on lesion-induced growth in thehippocampus. II. Hormone replacement. Exp Neurol 118:47–52[CrossRef][Medline]
  31. Stone DJ, Rozovsky I, MorganTE, Anderson CP, Finch CE 1998 Increased synaptic sprouting in response to estrogen via an apolipoprotein E-dependent mechanism: implications for Alzheimer’s disease. J Neurosci 18:3180–3185[Abstract/Free Full Text]
  32. Nathan BP, Chang KC, Bellosta S, Brisch E, Ge N, Mahley RW, Pitas RE 1995 The inhibitory effect of apolipoprotein E4 on neurite outgrowth is associated with microtubule depolymerization. J Biol Chem 270:19791–19799[Abstract/Free Full Text]
  33. Nathan BP, Jiang Y, Wong GK, Shen F, Brewer GJ, Struble RG 2002 Apolipoprotein E4 inhibits, and apolipoprotein E3 promotes neurite outgrowth in cultured adult mouse cortical neurons through the low-density lipoprotein receptor-related protein. Brain Res 928:96–105[CrossRef][Medline]
  34. Sun Y, Wu S, Bu G, Onifade MK, Patel SN, LaDu MJ, Fagan AM, Holtzman DM 1998 Glial fibrillary acidic protein-apolipoprotein E (apoE) transgenic mice: astrocyte-specific expression and differing biological effects of astrocyte-secreted apoE3 and apoE4 lipoproteins. J Neurosci 18:3261–3272[Abstract/Free Full Text]
  35. Arendt T, Schindler C, Bruckner MK, Eschrich K, Bigl V, Zedlick D, Marcova L 1997 Plastic neuronal remodeling is impaired in patients with Alzheimer’s disease carrying apolipoprotein {epsilon}4 allele. J Neurosci 17:516–529[Abstract/Free Full Text]
  36. Struble RG, Rosario ER, Kircher ML, Ludwig SM, McAdamis PJ, Watabe K, McAsey ME, Cady C, Nathan BP 2003 Regionally specific modulation of brain apolipoprotein E in the mouse during the estrous cycle and by exogenous 17ß estradiol. Exp Neurol 183:638–644[CrossRef][Medline]
  37. Levin-Allerhand J, McEwen BS, Lominska CE, Lubahn, DB, Korach, KS, Smith, JD 2001 Brain region-specific up-regulation of mouse apoliporotein E by pharmacological estrogen treatments. J Neurochem 79:796–803[CrossRef][Medline]
  38. Stone DJ, Rozovsky I, Morgan TE, Anderson CP, Hajian H, Finch CE 1997 Astrocytes and microglia respond to estrogen with increased apoE mRNA in vivo and in vitro. Exp Neurol 143:313–318[CrossRef][Medline]
  39. Rozovsky I, Hoving S, Anderson CP, O’Callaghan J, Finch CE 2002 Equine estrogens induce apolipoprotein E and glial fibrillary acidic protein in mixed cultures. Neurosci Lett 323:191–194[CrossRef][Medline]
  40. Brewer GJ 1997 Isolation and culture of adult rat hippocampal neurons. J Neurosci Methods 71:143–155[CrossRef][Medline]
  41. Demeester N, Castro G, Desrumaux C, De Geitere C, Fruchart JC, Santens P, Mulleners E, Engelborghs S, De Deyn PP, Vandekerckhove J, Rosseneu M, Labeur C 2000 Characterization and functional studies of lipoproteins, lipid transfer proteins, and lecithin:cholesterol acyltransferase in CSF of normal individuals and patients with Alzheimer’s disease. J Lipid Res 41:963–974[Abstract/Free Full Text]
  42. Walmer DK, Wrona MA, Hughes CL, Nelson KG 1992 Lactoferrin expression in the mouse reproductive tract during the natural estrous cycle: correlation with circulating estradiol and progesterone. Endocrinology 131:1458–1466[Abstract]
  43. Teter B, Xu PT, Gilbert JR, Roses AD, Galasko D, Cole GM 2002 Defective neuronal sprouting by human apolipoprotein E4 is a gain-of-negative function. J Neurosci Res 68:331–336[CrossRef][Medline]
  44. Brinton RD 2001 Cellular and molecular mechanisms of estrogen regulation of memory function neuroprotection against Alzheimer’s disease: recent insights and remaining challenges. Learn Mem 8:121–133[Abstract/Free Full Text]
  45. Boyles JK, Pitas RE, Wilson E, Mahley RW, Taylor JM 1985 Apolipoprotein E associated with astrocytic glia of the central nervous system and with nonmyelinating glia of the peripheral nervous system. J Clin Invest 76:1501–1513
  46. Pitas RE, Boyles JK, Lee SH, Foss D, Mahley RW 1987 Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins. Biochim Biophys Acta 917:48–61[Medline]
  47. Nathan BP, Nisar R, Randall S, Short J, Sherrow M, Wong GK, Struble, RG 2001 Time course of apolipoprotein E levels following olfactory nerve lesioning in mice. Exp Neurol 172:128–136[CrossRef][Medline]
  48. Slezak M, Pfrieger FW 2003 New roles for astrocytes: Regulation of CNS synaptogenesis. Trends Neurosci 26:531–535[CrossRef][Medline]
  49. Holtzman DM, Pitas RE, Kilbridge J, Nathan B, Mahley RW, Bu G, Schwartz AL 1995 Low density lipoprotein receptor-related protein mediates apolipoprotein E-dependent neurite outgrowth in a central nervous system-derived neuronal cell line. Proc Natl Acad Sci USA 92:9480–9484[Abstract/Free Full Text]
  50. Bellosta S, Nathan BP, Orth M, Dong LM, Mahley RW, Pitas RE 1995 Stable expression and secretion of apolipoproteins E3 and E4 in mouse neuroblastoma cells produces differential effects on neurite outgrowth. J Biol Chem 270:27063–27071[Abstract/Free Full Text]
  51. Saunders AM 2000 Apolipoprotein E and Alzheimer disease: an update on genetic and functional analyses. J Neuropathol Exp Neurol 59:751–758[Medline]
  52. Qiu Z, Crutcher KA, Hyman BT, Rebeck GW 2003 ApoE isoforms affect neuronal N-methyl-D-aspartate calcium responses and toxicity via receptor-mediated processes. Neuroscience 122:291–303[CrossRef][Medline]
  53. Buttini M, Akeefe H, Lin C, Mahley RW, Pitas RE, Wyss-Coray T, Mucke L 2000 Dominant negative effects of apolipoprotein E4 revealed in transgenic models of neurodegenerative disease. Neuroscience 97:207–210[CrossRef][Medline]
  54. Yaffe K, Haan M, Byers A, Tangen C, Kuller L 2000 Estrogen use, APOE, and cognitive decline: evidence of gene-environment interaction. Neurology 54:1949–1954[Abstract/Free Full Text]



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