Physical Interaction of ApoE with Amyloid Precursor Protein Independent of the Amyloid Abeta Region in Vitro*

Silke Habeta Dagger , Friedrich FresserDagger , Silvano KöchlDagger , Konrad Beyreuther§, Gerd UtermannDagger , and Gottfried BaierDagger

From the Dagger  Institute for Medical Biology and Human Genetics, University of Innsbruck, Schoepfstrasse 41, A-6020 Innsbruck, Austria and the § Center of Molecular Biology, University of Heidelberg, D-69120 Heidelberg, Germany

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

Variation at the APOE gene locus has been shown to affect the risk for Alzheimer's disease. To gain deeper insight into the postulated apoE-mediated amyloid formation, we have characterized the three common apoE isoforms (apoE2, apoE3, and apoE4) regarding their binding to amyloid precursor protein (APP). We employed the yeast two-hybrid system and co-immunoprecipitation experiments in cell culture supernatants of COS-1 cells, ectopically expressing apoE isoforms and APP751 holoprotein or a COOH-terminal Abeta deletion mutant protein, designated APPtrunc. We found that all three apoE isoforms were able to bind APP751 holoprotein in an Abeta -independent fashion. The interacting domains could be mapped to the NH2 termini of APP (amino acids 1-207) and apoE (amino acids 1-191). As a functional consequence of this novel APP751 ectodomain-mediated apoE binding, the secretion of soluble APP751 is differentially affected by distinct apoE isoforms in vitro, suggesting a new "chaperon-like" mechanism by which apoE isoforms may modulate APP metabolism and consequently the risk for Alzheimer's disease.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

Human apolipoprotein E (apoE),1 which is coded by a gene on chromosome 19q13.2, is a polymorphic protein and is known to play a major role in lipoprotein metabolism and cholesterol homeostasis in the brain (1-5). ApoE exists in three common genetic forms, designated apoE2, apoE3, and apoE4, which differ by single amino acid substitutions at one of two positions of the proteins 299-amino acids long primary structure (6, 7). Variation at the APOE gene locus has long been known to affect plasma cholesterol concentrations and subsequently the risk for atherosclerosis and coronary heart disease (1, 8). Recently it was realized that the apoE4 isoform is also associated with late onset sporadic and familial Alzheimer's disease (AD), the cause of dementia, effecting up to 5% of the population over age 65. Continuing gains in life expectancy have made AD by far the most common form of dementia worldwide (9-16). The effect of APOE4 appears to be dependent on gene dosage, i.e. homozygotes for the APOE4 allele tend to develop AD at an earlier age then heterozygotes (11, 17). Presence of an APOE4 allele is, however, neither necessary nor sufficient to develop AD (18). ApoE4 is therefore considered a susceptibility factor for AD. Interestingly, the apoE2 isoform seems to demonstrate a protective effect in AD, emphasizing the significant influence of the APOE genotype as a modifier of AD progression (17).

Although the genetic link between APOE and AD has been confirmed by many laboratories, the pathophysiological mechanism(s) by which the apoE4 isoform confers increased susceptibility and the apoE2 isoform confers decreased susceptibility to the disease is not yet understood. Quantitative neuropathological assessment reveals that amyloid deposits (19, 20) but not neurofibrillary tangles (19) are elevated in association with the apoE4 isoform, suggesting an influence of apoE primarily on amyloid formation in AD. Additionally, AD patients with the apoE4 isoform have more but not larger plaques than AD patients with the apoE3 isoform (21). Because amyloid Abeta peptide, a proteolytic product of the APP (22) and main component of the senile plaques, is hydrophobic, apoE molecules have been implicated in its aggregation and/or clearance (10, 23, 24). Differential binding of apoE isoforms to the amyloid Abeta peptide has been suggested as a mechanism. In our study the three common apoE isoforms, apoE2, apoE3, and apoE4, have been biochemically characterized regarding their differential binding to APP holoprotein in vitro.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Plasmids-- pAS2-1/apoE2, pAS2-1/apoE3, pAS2-1/apoE4, and pAS2-1/apoE3 NH2 (amino acids 1-191) were constructed by cloning the full-length or partial apoE cDNA (lacking the leader sequence) into the EcoRI/SalI sites of GAL4-DBD vector pAS2-1 using PCR and recombinant PCR primers apoE-SENSE: 5'-GGA TGC GAA TTC AAG GTG GAG CAA GCG GTG GA-3' and apoE-ANTISENSE: 5'-AGG CTT GTC GAC TCA GTG ATT GTC GCT GGG CAC-3' or apoE-NH2/1-191 ANTISENSE: 5'-GCC CAC GTC GAC CTA CCG CAC GCG GCC CTG TTC CA-3', respectively, engineered to create proper compatible ends and the correct reading frame for expression as fusion proteins in the context of the GAL4 DNA binding domain. Similarly, pACT2/APP751 were cloned into SmaI/SalI of pACT2, respectively, using APP-SENSE: 5'-GAC GGT CCC GGG GCT GGA GGT ACC CAC TGA T-3' and APP-ANTISENSE: 5'- GAT GAC GTC GAC TTC AGC TAT GAC AAC ACC GCC C-3'. pACT2/APPtrunc (amino acids 1-635) was constructed by COOH-terminal deletion using the endogenous BglII site, 5' of the Abeta sequence in the APP cDNA. pACT2/APP COOH-terminal deletion mutants, amino acids 1-342 and 1-207, have been constructed employing the endogenous restrictions sites XhoI and BbsI, respectively. The plasmids pEF-apoE2, apoE3, and apoE4 were constructed by shuffling the full-length coding regions (including the leader sequence) from pUC-apoE3 (obtained from J. Smith, Rockefeller University, NY), pCTV-apoE2, pCTV-apoE4 (obtained from K. Weisgraber, University of California San Francisco, CA), and apoE3-NH2 cDNA (amino acids 1-191, generated by PCR using recombinant PCR primers apoE-LEADER: 5'-CCA ATC TCT AGA GTC GAC ATG AAG GTT CTG TGG GCT GCG TTG-3' and apoE-NH2/1-191 ANTISENSE: 5'-GCC CAC ACT AGT CTA CCG CAC GCG GCC CTG TTC CA-3') into expression plasmid pEF-neo (a kind gift from Y. Liu, LIAI, CA) downstream and under the control of the elongation factor-1alpha promoter. Human APP full-length cDNA was loned into the HindIII/SalI sites of the CMV expression vector pTAG-CMVneo (25) employing PCR primers APP-Leader: 5'-GCC CCG AAG CTT GTC GCG ATG CTG CCC GGT TT-3' and APP-751-TAG-COOH: 5'-GCG GGG GTC GAC GTT CTG CAT CTG CTC AAA GAA CTT GT-3' to engineer a COOH-terminal fusion-TAG. Similarly, APP-trunc was cloned into pTAG-CMV using PCR primers APP-Leader (see above) and APPtrunc-TAG-COOH: 5'-CTT CAC GTC GAC GAT CTC CTC CGT CTT GAT ATT TG-3'. Correct constructs have been identified and confirmed by restriction analysis and partial DNA sequencing using vector-specific primers (5'-GAL4-DBD: 5'-CAT CGG AAG AGA GTA G-3', 3'-GAL4-DBD: 5'-CCT AAG AGT CAC TTT AAA A-3', 5'-GAL4-AD: 5'- TAC CAC TAC AAT GGA TG-3', 3'-GAL4-AD: 5'- ATA AAT GAA AGA AAT TGA GAT-3', 5'-EF-1alpha : 5'-TGG ATC TTG GTT CAT TCT CAA GCC-3', 5'-T7: 5'- TAA TAC GAC TCA CTA TA-3' and 3'-SP6: 5'- ATT TAG GTG ACA CTA TA-3').

Yeast Two-hybrid Screen-- The genotype of the Saccharomyces cerevisiae reporter strain HF7c used for the two-hybrid screening, is MATa, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4-542, gal80-538, LYS2::GAL1-HIS3, URA3:: (GAL4, 17-mers)3-CYC1-lacZ (CLONTECH). Stains were grown under standard conditions in rich or synthetic medium with appropriate supplements at 30 °C. For the yeast two-hybrid screening, DBD-apoE baits were co-transformed with the APP-AD fusion cDNA in the pACT vector (CLONTECH) into the HF7c yeast strain as described by the manufacturer, and the transformants were assayed for beta -galactosidase activity by transferring individual colonies on filters placed on selection medium. The plates were incubated for 2 days at 30 °C, the filters lifted and immersed in liquid nitrogen for 10 s. After thawing at room temperature, the filters were placed on filter circles saturated with X-gal solution in a Petri dish (permeabilized cells up) and incubated overnight. For quantitative analysis, beta -galactosidase reporter protein was measured in the cell extracts using a beta -galactosidase ELISA (5 Prime right-arrow 3 Prime, Inc., Boulder, CO). The results shown are obtained with different preparations of expression plasmids and represent the mean ± S.E. from four representative experiments done in triplicate.

Immunoblotting of GAL4-DBD Fusion Baits in Yeast Extracts-- 5 ml of transformed yeast cells grown overnight in selective medium lacking tryptophan were used to inoculate 15 ml of YPD medium. At an optical density (600 nm) of 0.5, the cells were pelleted, washed, resuspended at 5 × 108 cells/ml in ice-cold radioimmune precipitation buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 µg/ml aprotinin/leupeptin, and 1 mM phenylmethylsulfonyl fluoride) and frozen at -20 °C. Samples were analyzed by SDS-polyacrylamide gel electrophoresis (10%), transferred to polyvinylidene difluoride membrane (Millipore, Vienna), and fusion proteins were detected using a GAL4-DBD (Santa Cruz Biotechnology, Inc.) or GAL4-AD (Upstate Biotechnology) specific antibodies, followed by a rabbit anti-mouse IgG-peroxidase conjugate and a chemiluminescence detection kit (Pierce).

Transient Transfection of COS-1 Cells-- The SV40-transformed African green monkey kidney cell line COS-1 was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured as recommended by ATCC. For transient transfections, cells were seeded at a density of 2 × 106 cells/well in 10-cm Petri plates in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C. One day later, the cells were transfected with 20 µg of circular plasmid DNA/dish by electroporation (BTX, San Diego, CA) with the following settings: 100 V, 48 Ohm and 1275 µF, or by LipofectAMINETM in Opti-MEM medium (Life Technologies, Inc.) as described by the manufacturer. 24 h post-transfection, cells were lysed in lysis buffer (5 mM NaP2P, 5 mM NaF, 5 mM EDTA, 50 mM NaCl, 50 mM Tris, pH 7.3, 2% Triton X-100, 50 µg/ml each aprotinin and leupeptin). Similarly, cell culture media were harvested by precipitation in 5% trichloroacetic acid.

Co-immunoprecipitation-- Cell culture media were precleared by incubating with 50 µl of protein G-Sepharose beads for 2 h at 4 °C and then immunoprecipitated overnight at 4 °C with the indicated antibodies at 5 µg/ml final antibody concentration followed by addition of 50 µl of protein G-Sepharose beads for the last 2 h. Immunoprecipitates were collected by centrifugation for 1 min at 13,000 rpm and 4 °C in an Eppendorf microfuge and washed six times with phosphate-buffered saline, 0.02% Tween buffer. Immuno-precipitates were resuspended in SDS-polyacrylamide gel electrophoresis sample buffer, boiled for 5 min at 95 °C and separated on 10% Tris-glycine gels (Novex). Immunoblot analysis using apoE (Calbiochem), APP (Boehringer Mannheim 22C11 or WO-2, obtained from K. Beyreuther, Heidelberg) and TAG-specific (H902, Ref. 25) antibodies was performed.

Metabolic Labeling and Immunoprecipitation-- COS-1 cells transfected with apoE and APP expression plasmids were treated with minimum essential medium lacking methionine, supplemented with 100 µCi of [35S]methionine (Amersham Pharmacia Biotech, Braunschweig, Germany) for 1 h. For pulse-chase analysis, the chase was performed with 1 mM L-methionine in minimum essential medium for various periods of time as indicated before standard cell lysis and immunoprecipitation analysis of APP holoprotein employing mAb WO-2 (anti-Abeta 1-16).

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

In our study the three common apoE isoforms, apoE2, apoE3, and apoE4, have been biochemically characterized regarding their binding to APP in vitro. In contrast to previous studies employing synthetic Abeta peptides (10, 23, 24), we investigated the role of the Abeta region in a potential direct interaction of apoE and APP holoprotein, initially employing the GAL4 two-hybrid system. As result, a specific and direct physical association of apoE and APP holoprotein could be demonstrated (Fig. 1A). Additionally and to our surprise, an Abeta -independent interaction of apoE and APPtrunc, a COOH-terminal deletion mutant of APP (amino acids 1-635) devoid of the Abeta region, was observed (Fig. 1B). Interaction results were strictly dependent on the combined presence of one of the GAL4-apoE isoform baits plus one of the GAL4-APP ligands. Specificity controls employing GAL4-p53 and GAL4-SV40 ligands or empty vector in exchange of either apoE or APP and APPtrunc did not activate the beta -galactosidase expression (data not shown).


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Fig. 1.   Direct physical interaction of distinct apoE isoforms with APP751 holoprotein (A) and APP751 Abeta deletion mutant APPtrunc (B) in the yeast two-hybrid system. Human apoE isoforms and APP holoprotein or APPtrunc, a COOH-terminal Abeta deletion mutant cDNA, were cloned into pAS2-1 or pACT2 (CLONTECH) as a fusion protein in the context of the GAL4 DNA binding or GAL4 transcription activation domain, respectively. Recombinant plasmid constructs were confirmed by DNA sequencing. Expression studies of the recombinant GAL4 fusion proteins in the yeast H7Fc host cells (see immunoblot, panels C and D) demonstrated GAL4-apoE and GAL4-APP fusion proteins (as detected by immunoblotting using mAbs specific for GAL4-DBD or GAL4-AD, respectively) as protein doublet with the expected molecular weight of approx 45 and approx 140 kDa. Subsequently, the GAL4-DBD hybrid constructs pAS-apoE2, -apoE3, and -apoE4 and GAL4-AD hybrid constructs pACT-APP holoprotein or COOH-terminal truncation mutant APPtrunc together with pAS or pACT vector control were transformed into the yeast strain H7Fc as indicated. Protein-protein binding was detected by quantitative ELISA analysis of the GAL4 reporter protein beta -galactosidase. The results shown were obtained with different preparations of expression plasmids and represent the mean ± S.E. from four experiments, each done in triplicate.

To biochemically confirm the relevance of this putative apoE and APP751 interaction, we used COS-1 cells, which do not express apoE, for transient transfection with expression plasmids encoding distinct apoE isoforms and APP751 holoprotein or APPtrunc, the COOH-terminal Abeta deletion mutant, respectively (Fig. 2) First, overexpressed and, due to endoproteolytic processing, soluble (s)APP751 was immunoprecipitated from cell culture media, and the resultant precipitates, e.g. apoE-APP complexes, were detected by immunoblotting with antisera specific to apoE or APP. As shown in Fig. 2A, apoE specifically co-immunoprecipitates with sAPP751, indicating an interaction of these two proteins. In another set of experiments, ectopically expressed apoE isoforms (and apoE-associated APPtrunc) were immunoprecipitated from cell culture media, and the resultant precipitates were analyzed by immunoblotting with either mAb alpha TAG, specific for the epitope-TAG engineered into the recombinant sAPPtrunc (see cartoon in Fig. 2C) or anti-apoE antibodies. Consistently with the apoE-APP interaction detected in the yeast two-hybrid results, a specific and Abeta -independent interaction of apoE and sAPP could be demonstrated in these co-immunoprecipitation assays (Fig. 2B). Again, all three apoE isoforms were able to form complexes with sAPP751. Since the APPtrunc used for transfections lacks the Abeta region, the findings of Fig. 2B demonstrate that the observed apoE-APP interaction requires domains of APP upstream of the Abeta region. Recently, a physical interaction of sAPP and baculovirus-expressed apoE3 and apoE4 has been reported (26), an observation consistent with our co-expression studies reported here.


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Fig. 2.   Interaction of distinct apoE isoforms with APP751 holoprotein (A) and APP751 Abeta deletion mutant APPtrunc (B) in co-immunoprecipitation assays in vitro. An equal number of COS-1 cells have been transfected with distinct apoE isoform pEF1alpha -neo expression plasmids (pEF-apoE2, -apoE3, and -apoE4 or empty vector control) together with either pCMV-APP holoprotein (A, +) or pTAG/CMV-APPtrunc (B, +) or empty vector control (A and B, -) as indicated. Following transfection, co-immunoprecipitation assays were performed from COS-1 cell culture media. Immunoprecipitates were resolved by 12% Tris-glycine gel electrophoresis and detected by immunoblot analysis using alpha (apoE), alpha (APP) 22C11, or alpha (TAG), respectively, to detect binding of apoE with sAPP proteins. To distinguish between Abeta -containing endogenously expressed sAPP and our recombinant sAPPtruncTAG, a COOH-terminal Abeta deletion mutant of APP (amino acids 1-635), a TAG-epitope plus the proper mAb alpha (TAG) for immunodetection has been employed (B). Immunoprecipitation and immunodetection of sAPP with APP-specific antibodies (A, upper panel) and apoE with the apoE-specific antibodies (B, lower panel) served as positive control. The band seen at 50 kDa represents the reduced IgG heavy chain (HC). Co-immunoprecipitation of apoE3 without sAPP751 overexpression (A, lane E3/-) could be observed after long exposure of the blot due to sAPP endogenously expressed from COS-1 cells. C, the cartoon outlining the structure of APP holoprotein versus APPtruncTAG. The results shown are representative data from three independent experiments.

To define the critical domains in the apoE-APP interaction, APP and apoE3 COOH-terminal deletion mutants have been employed in the yeast two-hybrid system. As shown in Fig. 3A, the initial definition of the binding pockets could be assigned to the NH2 terminus (amino acids 1-207) of APP and the NH2 terminus (amino acids 1-191) of apoE. This has been independently demonstrated in co-immunoprecipitation assays of ectopically expressed apoE and APP COOH-terminal deletion mutants (Fig. 3B), confirming the binding of sAPPtrunc to the NH2 terminus (amino acids 1-191) of apoE3.


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Fig. 3.   Critical domains in the apoE-APP interaction. A, APP and apoE3 COOH-terminal deletion mutants have been employed in the yeast two-hybrid system, assigning the interaction to the NH2 terminus (amino acids 1-207) of APP and the NH2 terminus (amino acids 1-191) of apoE. The GAL4-DBD hybrid constructs pAS-apoE3, pAS-apoE3-NH2 (amino acids 1-191) and distinct GAL4-AD hybrid pACT-APP-751 COOH-terminal truncation mutants were transformed into the yeast strain H7Fc as indicated. Specific protein-protein binding was detected by quantitative ELISA analysis of the GAL4 reporter protein beta -galactosidase. Interaction is marked as the means ± S.E. Specificity controls employing GAL4-p53 and GAL4-SV40 ligands or empty vector in exchange for either apoE or APP did not activate the beta -galactosidase expression (data not shown). B, co-immunoprecipitation (Co-IP) assays of ectopically expressed apoE and APP COOH-terminal deletion mutants. Following transfection, co-immunoprecipitation assays were performed from COS-1 cell culture media. Immunoprecipitates were resolved by 12% Tris-glycine gel electrophoresis and detected by immunoblot analysis using alpha (TAG) and alpha (apoE), respectively, to detect binding of apoE NH2 terminus (amino acids 1-191) with the recombinant sAPPtruncTAG protein. Nonspecific binding of APPtrunc to the protein G-Sepharose beads has not been observed (see Fig. 2B, lane V/+).

To investigate whether this novel interaction may affect APP processing, we used COS-1 cells for transient co-transfection with apoE2, apoE3, apoE4, and APP holoprotein. 24 h post-transfection, COS-1 cell culture media were investigated for apoE and sAPP protein concentrations. As shown in Fig. 4A, a significant apoE isoform-dependent inhibition of sAPP751 secretion could be observed. ApoE2 demonstrated the strongest effect on sAPP followed by apoE3 and apoE4. Reduced concentrations of the soluble form of APP in the cellular supernatant may be explained by intracellular retention of APP. This was indeed demonstrated by the apoE-mediated accumulation of mature APP in the Triton X-100 soluble cellular fraction (Fig. 4B). No significant amount of APP could be detected in the detergent-resistant fraction (not shown). Similar effects could be seen with APP695 (data not shown). ApoE isoforms therefore influence secretion and/or retro-endocytosis of APP from transfected COS-1 cells in an isoform-specific manner. Again, this apoE isoform-specific effect on sAPP751 was Abeta -independent since they were also observed with APPtrunc (Fig. 4C).


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Fig. 4.   ApoE isoform-specific retention of APP751 holoprotein (A and B) and APP751 Abeta deletion mutant APPtrunc (C) in co-expression studies. COS-1 cells were transiently transfected with either of the three apoE isoforms or empty vector control and APP751 holoprotein (A and B) and APPtrunc (C) as indicated. Relative concentrations of apoE isoforms and APP751 holoprotein or APPtrunc, a COOH-terminal Abeta deletion mutant of APP, in transfected COS-1 supernatants (TCA-Medium) versus Triton X-100 cellular lysates (Triton Cell-Lysat) have been detected by immunoblotting using mAb alpha (APP) 22C11 (A and B) or alpha TAG (C), respectively. Similar effects could be seen with APP695 (data not shown). The results shown were obtained with different preparations of expression plasmids. Statistical analysis of the experiments described was determined by densitometric quantification. The bar marked with the asterisk was set at 100% for calculation purposes, and data are expressed as the means ± S.E. of at least three independent experiments.

To further distinguish between apoE-mediated retention of sAPP or retro-endocytosis of sAPP, pulse-chase experiments have been performed over chase periods between 30 and 150 min. A representative result is shown in Fig. 5 demonstrating, only in the presence of apoE2, a significant intracellular accumulation of the APP holoprotein up to 90 min. Similar albeit reduced retention effects on APP holoprotein could be observed with apoE3 and apoE4 (data not shown). Due to the time frame of intracellular APP accumulation, apoE isoforms seem to mediate retention of APP mainly by reducing its secretion rate, an observation consistent with the postulated chaperon-like function of intracellular apoE. Additionally, however, apoE-mediated APP retro-endocytosis may also contribute to the observed intracellular APP accumulation. Undoubtedly, more work is necessary to determine the precise mechanism utilized by apoE isoforms, but our data represent an important step toward the identification and characterization of this novel apoE function involved in modulation of APP metabolism.


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Fig. 5.   ApoE2-mediated intracellular retention of APP751 holoprotein in co-expression studies. COS-1 cells were transiently transfected with either apoE2 or empty vector control and APP751 holoprotein as indicated. For immunoprecipitation of metabolically labeled APP751 holoprotein (1-h labeling time) mAb alpha (APP) WO-2 was used. Relative concentrations of APP751 holoprotein in Triton X-100 cellular lysates (Triton Cell-Lysat) have been detected by pulse-chase experiments at 30-, 60-, 90-, 120-, and 150-min chase periods as indicated and followed by SDS-polyacrylamide gel electrophoresis and autoradiography.

Consistently, we found that the effect of apoE/APP751 interaction is bilateral, since APP751 prevents apoE secretion to the culture medium (Fig. 6A) and furthermore leads to an apparent reduction of intracellular apoE (Fig. 6B). This appears to be mediated in part by the ubiquitin-proteasome pathway, since N-acetyl-leucyl-leucyl-norleucinal, a potent inhibitor of this degradation pathway, can block apoE degradation by approx 30% (data not shown). The effects shown in Figs. 4-6 are not due to different expression levels of apoE2, apoE3, and apoE4 in transfected COS-1 cells (Fig. 6, see for control apoE the lanes lacking APP (-)). Additionally, no similar apoE effect was observed with other glycoproteins besides APP, e.g. beta -2 glycoprotein I secretion was not inhibited by apoE2 co-expression (data not shown). This further indicates a physiological association of apoE isoforms with APP, suggesting a potential chaperon-like function of apoE isoforms in APP processing in the order apoE2>> apoE3approx apoE4.


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Fig. 6.   APP-dependent retention of apoE isoforms in co-expression studies. COS-1 cells were transiently transfected with either of the three apoE isoforms and APP751 holoprotein (+) or empty vector control (-) as indicated. Immunodetection for the presence of apoE in transfected COS-1 supernatants (A, TCA-Medium) versus Triton X-100 cellular lysates (B, Triton Cell-Lysat) was performed using alpha (apoE) antibodies. As expression control, COS-1 cells were single-transfected with either of the three apoE isoforms or empty vector controls as indicated. The results shown were obtained with different preparations of expression plasmids. The bar marked with the asterisk was set at 100% for calculation purposes. Densitometric quantification is expressed as the means ± S.E. of at least three independent experiments.

In conclusion, apoE was demonstrated to directly bind via its NH2 terminus to APP holoprotein within the ectodomain, independent of the Abeta region in vitro. This was independently shown by the yeast two-hybrid system and by co-immunoprecipitation assays from ectopically transfected COS-1 cell culture media. Co-expressed apoE affects sAPP secretion in an apoE isoform-specific way in vitro. This novel apoE-APP ectodomain interaction may reflect a new potential physiological role of distinct apoE isoforms in APP metabolism, e.g. in astrocytes and microglia cells, endogenously co-expressing apoE (27, 28) and APP (29, 30). This may affect either Abeta formation or alternatively may interfere with APP-mediated signal transduction pathways (31, 32). The relevance for the in vivo situation, however, remains to be shown.

    ACKNOWLEDGEMENTS

We are grateful to Dr. V. Zannis (Boston) for helpful discussions and to Dr. E. Preuss (Innsbruck), PDL, for the art work.

    FOOTNOTES

* This work was supported by Grants from the Austrian Ministry of Science and the European Communities/Biomed 2 Program (BMH4-CT96-0898).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. Tel.: 43-512-507-3451; Fax: 43-512-507-2861; E-mail: Gottfried.Baier{at}uibk.ac.at.

1 The abbreviations used are: apoE, apolipoprotein E; AD, Alzheimer's disease; PCR, polymerase chain reaction; X-gal, 5-bromo-4-chloro-3-indoyl beta -D-galactopyranoside; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody.

    REFERENCES
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Abstract
Introduction
Procedures
Results & Discussion
References

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