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Annals of Clinical & Laboratory Science 32:65-74 (2002)
© 2002 Association of Clinical Scientists

Isoform-specific Effect of Apolipoprotein E on Endocytosis of ß-Amyloid in Cultures of Neuroblastoma Cells

Kazuyoshi Yamauchi1, Minoru Tozuka1, Hiroya Hidaka1, Tetsuo Nakabayashi1, Mitsutoshi Sugano1 and Tsutomu Katsuyama2
1 Central Clinical Laboratories, Shinshu University Hospital, Matsumoto, Japan
2 Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto, Japan

Address correspondence to Kazuyoshi Yamauchi, Ph.D., Central Clinical Laboratories, Shinshu University Hospital, 3-1-1 Asahi, Matsumoto 390-8621, Japan; tel 81 263 37 2802; fax 81 263 34 5316; e-mail yamauchi{at}hsp.md.shinshu-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Investigation of the interactions of nerve cells with human apolipoprotein E (apoE), ß-amyloid (Aß), and their complex, which are known to be included in senile plaques, is necessary to clarify the functional role of apoE in the pathogenesis of Alzheimer’s disease. Using flow cytometric analysis, we investigated the isoform-specific effects of apoE on the endocytosis of Aß in cultured neuroblastoma cells. The level of internalized Aß within the cells was dependent on the culture time and the kind of apoE isoform present. Both apoE3 and apoE4 enhanced the internalization of Aß; however, no difference was observed between their effects. The internalized Aß was hardly catabolized at all in the presence of apoE4, while rapid clearance of Aß was observed in the presence of apoE3. Unlike apoE3 and apoE4, apoE2 had no effect on Aß clearance from the media. The isoform-specific effects of apoE on the endocytosis of Aß may be implicated in the development of Alzheimer’s disease, and if so, each isoform of apoE would induce a different incidence of that disease.

(received 2 October 2001; accepted 15 October 2001)

Keywords: apo E, Alzheimer’s disease, ß-amyloid, senile plaques, flow cytometry, neuroblastoma cells

Abbreviations: apoE, apolipoprotein E; Aß, ß-amyloid; AD, Alzheimer’s disease; CNS, central nervous system; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescent isothiocyanate; RAGE, receptor for advanced glycation end-products; LDL, low density lipoprotein; VLDL, very low density lipoprotein


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Alzheimer’s disease (AD), a representative cause of senile dementia, is characterized by the presence of extracellular senile plaques within brain tissue [1–3]. The primary component of this hallmark is generated by a splicing of the ß-amyloid precursor protein [4,5]. Previous studies demonstrated that apolipoprotein E (apoE) accumulates as a complex with Aß in this amyloid deposit, and suggested that apoE is implicated in the development of AD [1,3,6–8]. ApoE, a protein consisting of 299 amino acids with molecular mass of 35 kDa, is involved in cholesterol transport and metabolism as a component of lipoproteins [9,10]. Within the central nervous system (CNS), the second most active tissue in terms of apoE production [11,12], apoE is mainly produced in astrocytes and microglia [13,14]. ApoE has 3 major isoforms [E2 (Cys112, Cys158), E3 (Cys112, Arg158), and E4 (Arg112, Arg158)], the products of 3 independent alleles at a single genetic locus [9,15,16]. Thus, 3 homozygous (apo-E2/E2, E3/E3, and E4/E4) and 3 heterozygous phenotypes (apo E3/E2, E4/E3, and E4/E2) are represented within the population.

The polymorphism of apoE influences the development of AD; indeed, its incidence is significantly greater in subjects who have apoE4 [3,7,17,18]. Several possibilities have been proposed to explain this correlation. In particular, an isoform-specific interaction of apoE with Aß during the formation of senile plaques has been highlighted. This may be implicated in the physiological mechanism that underlies the participation of apoE in the development of AD. However an actual causal relationship between apoE and the development of AD remains to be identified.

Previous studies have focused on the isoform-specific binding of apoE to Aß, and the cytotoxicity of the apoE-Aß complex [3,19–23]. Investigation of the isoform-specific role of apoE in the movement of Aß within the CNS is essential if we are to clarify the mechanism for the development of AD. To date, only a few studies have been carried out on the movement of Aß and its complex with apoE within neuronal cells. The authors of these studies had difficulties in quantitative analyses, because of the methodological limitations of immunohistochemical and immunoblot methods [24–27]. To perform a quantitative investigation, we established a simple and sensitive method using flow cytometry, and we used it to assess the isoform-specific effects of apoE on internalization of Aß and its complexes with apoE in cultures of neuroblastoma cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials.  Recombinant apoE2, apoE3, and apoE4 were purchased from Chemicon International Inc. (Temecula, CA). Aß1–42 peptide was purchased from Alexis Co. (San Diego, CA). Anti-apoE antibody (rabbit), anti-Aß antibody (rabbit), fluorescent isothiocyanate (FITC)-conjugated anti-rabbit IgG (goat), and horseradish-peroxidase-conjugated anti-rabbit IgG (goat) were purchased from Dako Co. (Glostrup, Denmark), IBL Co. (Gunma, Japan), Immunotech (Marseilles, France), and MBL Co. (Nagoya, Japan), respectively.

Cell line.  OAN cells [28], a gift from Garrett M. Brodeur, were maintained in RPMI Medium 1640 (Life Technologies, Inc., Rockville, MD), with 1% Antibiotic Antimycotic (Sigma Co., St. Louis, MO) and 10% fetal bovine serum (FBS) (Life Technologies, Inc., Rockville, MD), in 5% CO2-air at 37°C.

Preparation of conditioned media.  Each recombinant apoE (100 µg) was dissolved in 100 µl of 5 mol/L guanidine-HCl, to which was added 400 µl of phosphate-buffered saline (PBS), pH 7.4, and this was dialyzed against PBS. After dialysis, the solution volume was adjusted to 1 ml using PBS. To confirm the refolding success, the dissolved apoE was compared with cerebrospinal fluid (CSF) apoE by SDS-PAGE analysis. Aß1–42 peptide (500 µg) was dissolved in 20 µl of dimethyl sulfoxide (DMSO), to which was added 980 µl of PBS. The solutions of apoE and Aß were filtered using a 0.22 µm membrane followed by mixing and incubation for 24 hr at 37°C. After the incubation, the mixture was added to serum-free RPMI Medium 1640 containing 1% Antibiotic Antimycotic. The final concentrations of apoE and Aß were adjusted to 5.4 mg/L and 10.0 mg/L, respectively. The final DMSO concentration was 0.2%, which was shown not to affect to the results in such studies [25].

Cell culture.  Cells (1 x 104/ml) were plated in RPMI Medium 1640 plus 1% Antibiotic Antimycotic containing 10% FBS, and were grown on 100 mm dishes at 37°C in a 5% CO2 humidified atmosphere. After 4 or 5 days in culture, the media were aspirated, and the cells adhering to the bottom of the dish were washed twice with PBS. Then, the prepared conditioned media were added and cultures were allowed to proceed under the conditions described above.

FITC-labeled apoE.  Each recombinant apoE was dissolved at 200 mg/L in 0.5 mol/L carbonate buffer (pH 9.5), then mixed with FITC (Sigma, St. Louis, MO) in a ratio of 1:50 (w/w). Following incubation and stirring for 1 hr at room temperature, the FITC-labeled apoE was separated from excess unconjugated FITC by dialysis using PBS, pH 7.4. The integrity of the labeled apoE was confirmed by gel filtration chromatography, a Superose-6 column (Pharmacia Biotech, Uppsala, Sweden) equilibrated with PBS, pH 7.4, based on the criterion that a peak absorbing at 280 nm is consistent with a peak of fluorescence for FITC.

Flow cytometric analysis for Aß.  After conditioned media had been removed by aspiration, cultured cells were treated with enzyme-free cell-dissociation solution (‘’Hank’s based’’; Specialty Media, Inc., NJ) for 5 min at 37°C, and then centrifuged for 10 min at 1,000 rpm. After the supernatant had been removed by aspiration, the precipitated cells were washed with PBS twice and re-suspended in 200 µl of PBS. Then cells were fixed with 20% formalin neutralized buffer solution (Wako Pure Chemicals, Osaka, Japan), followed by permeabilizing with 0.5% saponin and 0.5% bovine serum albumin (BSA) in PBS containing 0.1% NaN3 for 10 min at room temperature. The non-specific binding sites were blocked by the incubation with 10-fold volume of PBS containing 10% goat serum (Dako Co., Glostrup, Denmark) (blocking buffer). We then added 100 µl of a 2-fold dilution of anti-Aß antibody in PBS containing 1% BSA to both aliquots. After 60-min incubation on iced water followed by 3 washes with PBS, we added 100 µl of a 100-fold dilution of FITC-conjugated anti-rabbit IgG, and incubation on iced water was allowed to proceed for 60 min. After 3 washes with PBS, the cells were stored on iced water prior to analysis by FACScan (Becton-Dickinson Co., Rutherford, NJ). All analyses were performed within 1 hr.

Flow cytometric analysis for apoE.  To assess the movement of FITC labeled-apoE, the dissociated cells, as described above, were divided into 2 aliquots for the separate analysis of extracellular (bound) and intracellular (internalized) apoE. For the former, one was simply incubated (for 15 min on iced water) with 10-fold volume of the blocking buffer. For the latter, the other was permeabilized by the method described above. After 3 washes with PBS, the analyses were performed using FACScan.

Fluorescence microscopic analysis.  Cultured cells on glass coverslips were fixed in 20% formalin neutralized buffer solution for 15 min at room temperature. After removing the fixed solution and washing twice with PBS, the cells were permeabilized as described above. We then added 100 µl of a 5-fold dilution of anti-Aß antibody in PBS containing 1% BSA, and incubated the mixture for 30 min at room temperature. After washing with PBS 3 times, we added 100 µl of a 100-fold dilution of FITC-conjugated anti-rabbit IgG, and allowed incubation to proceed for 30 min at room temperature. We then washed the coverslips with PBS 3 times, mounted the cells in glycerin, and observed them under a fluorescence microscope.

Determination of Aß.  Concentrations of Aß1–42 in the various media were determined by enzyme-linked immunosorbent assay (ELISA) [29]. Briefly, 25 µl of each medium was added to the microtiter plate, which was coated with anti-Aß1–42 monoclonal antibody, and this was incubated with 75 µl of a biotinylated anti-Aß1–42 monoclonal antibody for 1 hr at room temperature. After washing the plates 5 times with PBS containing 0.1% Tween 20, we added 100 µl of peroxidase-labeled streptavidin and incubated the mixture for 30 min at room temperature. We washed the plates 5 times and then added 100 µl of tetramethylbenzidine dihydrochloride. After 30-min incubation at room temperature, the reaction was stopped by adding 50 µl of 0.4 mol/L sulfuric acid, and the absorbance at 450 nm was measured using an ELISA processor II (Behringwerke AG, Marburg, Germany). A calibration curve was constructed by plotting the absorbance values obtained for Aß1–42 standard solutions (0, 125, 250, 500, 1000, 1500, 2000 pg/ ml), and the Aß1–42 concentrations in the various media were read from the curve. Each assay was carried out in triplicate.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Fig. 1Go shows histograms of fluorescence intensities obtained from neuroblastoma cells cultured in the presence or absence of Aß plus apoE4 by flow cytometric analysis. The internalized Aß was visualized by a reaction of the cells with anti-Aß antibody followed by the FITC-labeled second antibody. No cross-reactivity between anti-Aß antibody and apoE or the proteins in permeabilized neuroblastoma cells was observed by immunoblot analysis (data not shown). The mean channel of fluorescence intensity for the internalized Aß in cells not treated with Aß plus apoE (as background) was 20.15±4.54 (Fig. 1AGo). When cells were cultured in medium containing Aß and apoE, new peaks with higher fluorescence intensities were observed in the histograms, the mean channels of fluorescence intensities being raised to 63.64±7.83 (Fig. 1BGo). We confirmed the states of neuroblastoma cells supplied to the flow cytometric analysis by fluorescence microscopy.



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  		Fig. 1. Optimized flow cytometric analysis and immunofluorescence staining for intracellular Aß. Recombinant apoE4 and Aß1–42 were incubated for 24 hr at 37°C. After incubation, the mixture was added to serum-free RPMI Medium 1640 (final concentrations of 5.4 mg/L for apoE and 10.0 mg/L for Aß). Neuroblastoma cells were cultured in serum-free RPMI Medium 1640 containing (A) or not containing (B) the mixture of apoE4 and Aß, and analyzed using a flow cytometer and immunofluorescence staining with the anti-Aß antibody as described in Materials and Methods. The vertical and horizontal axies show the number of counted cells and the fluorescence intensity, respectively. Scale bar in the immunofluorescence staining pattern shows 20 µm.

 
To investigate the isoform-specific effects of apoE on the interaction between Aß and neuroblastoma cells, we prepared 3 types of media containing various incubation mixtures (Aß with apoE2, apoE3, and apoE4, respectively). The time-related changes in both fluorescent intensities for intracellular Aß and the concentrations of Aß in each culture medium are shown in Fig. 2Go.



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  		Fig. 2. Effect of apoE isoforms on the time-related changes in both fluorescence intensity for intracellular Aß and Aß concentration in the various conditioned media. An incubated mixture containing one of the recombinant apoE isoforms and Aß1–42 was added to serum-free RPMI Medium 1640 to give final Aß and apoE concentrations of 10.0 mg/L and 5.4 mg/L, respectively. Neuroblastoma cells were cultured in the various conditioned media (A, Aß1–42 alone; B, apoE2 and Aß1–42; C, apoE3 and Aß1–42; D, apoE4 and Aß1–42) for 1, 3, 6, and 9 hr, and then analyzed for intracellular Aß (solid circles) and Aß concentrations in each conditioned medium (open circles) using a flow cytometer and an ELISA assay, respectively. The values are mean±SD, and were derived from triplicate determinations in each of 3 separate experiments. They are expressed relative to the value for control cells, which were cultured in the presence of each apoE isoform alone.

 
Observation of the interaction between Aß and neuroblastoma cells in the presence of apoE was begun immediately after the start of culturing in the conditioned media and continued for 9 hr. Only small amounts of intracellular Aß were observed in the medium without apoE (Fig. 2AGo). The intracellular Aß level in cells cultured in medium containing apoE2 was similar to that in the medium containing Aß alone (Fig. 2BGo). Interestingly, a significant difference was observed between apoE3-and apoE4-containing media. In the former, the intracellular Aß reached a peak after 3 hr and then showed a tendency to decline time-dependently. In the later, the intracellular Aß reached a peak after 6 hr and showed no sign of significant attenuation after that. On the other hand, the reduction of Aß in the media containing apoE3 or apoE4 was maximal after 6 hr and remained constant until 9 hr. In contrast, Aß in the media containing apoE2, as well as Aß alone, slowly decreased in a time-dependent manner.

The percentage reductions in the amount of Aß in the various media after 9 hr in culture were as follows: 32.8±6.8% for Aß alone; 12.4±7.0% for Aß plus apoE2; 63.0±3.3% for Aß plus apoE3; and 68.3±5.2% for Aß plus apoE4. No significant difference was observed in these effects between apoE3 and apoE4; however, the percent reductions were significantly lower for Aß plus apoE2 than for Aß with either apoE3 or apoE4 (p <0.003). In addition, the percent reduction for Aß plus apoE2 was significantly lower than that for Aß alone (p <0.03).

A fluorescence microscopic analysis was also performed on the neuroblastoma cells after 0, 3, and Recombinant apoE isoforms were labeled with fluorescent isothiocyanate as described in Materials and Methods. The labeled apoE isoforms were incubated withAß1–42,and added to serum-free medium to give apoE and Aß1–42 final concentrations of 5.4 and 10.0 mg/L, respectively. Neuroblastoma cells were cultured in each conditioned medium for 6 hr, and then analyzed by flow cytometry. The valuse are means±SD, derived from duplicate determinations in each of 2 separate experiments. They are expressed relative to the value for untreated cells. Statistical significance was determined by Student’s t-test. 6 hr in culture under the various conditions described above (Fig. 3Go). As shown by flow cytometric analysis, the cells after 3 hr in culture in the Aß plus apoE3 or apoE4 containing medium showed strong fluorescence intensity for intracellular Aß. After 6 hr in culture, the fluorescence intensity of cells in apoE3-containing medium was attenuated, while that in apoE4-containing medium was unchanged. In contrast, apoE2 gave rise to the weakest fluorescence intensity for the Aß internalized within the cells throughout cell culture.



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  		Fig. 3. Immunofluorescence staining of neuroblastoma cells cultured in various conditioned media. The incubated mixtures of each recombinant apoE isoform and Aß1–42 were added to serum-free RPMI Medium 1640 to give final Aß and apoE concentrations of 10.0 mg/L and 5.4 mg/L, respectively. Neuroblastoma cells on grass coverslips were cultured in these condition media (A, Aß1–42 alone; B, apoE2 and Aß1–42; C, apoE3 and Aß1–42; D, apoE4 and Aß1–42) for 0, 3, and 6 hr. The cells were permeabilized followed by immunostaining for intracellular Aß with anti-Aß antibody. Scale bar, 20 µm.

 
Finally, the movement of apoE was determined by flow cytometry using FITC-labeled apoE (Table 1Go). The binding of FITC-labeled apoE to cells cultured with apoE3- or apoE4-containing medium was at approximately the same, regardless of whether Aß was present in the medium. A small amount of internalization was observed in apoE2-containing medium with or without Aß, but higher internalization levels were shown in cultures with apoE3 or apoE4; both of these were increased by the presence of Aß in the medium.


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  		Table 1. Relative levels of FITC-labeled apoE.
 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Recently, Bales et al [30] reported that Aß deposition is dramatically reduced in apoE-knockout mice. This implies that apoE is closely implicated in the formation of senile plaques. However, an actual examination of the movement of apoE, Aß, and their complexes, which are deposited in the AD brain in the form of senile plaques, is necessary if we are to clarify the role of apoE in the pathogenesis of AD.

To investigate the isoform-specific effects of apoE on the interaction between Aß and nerve cells, we used flow cytometric analysis to examine the movement (internalization and clearance) of Aß in the separate presence of each apoE isoform in cultured neuroblastoma cells. Usually, the movement of Aß is characterized by immunohistochemical and immunoblot analysis [24–27]; however, those methods have methodological limitations for producing quantitative data. The present study on neuroblastoma cell lines has shown that the fluorescence signal from internalization of Aß can be clearly discerned from the background, and that the specific Aß signal can be semi-quantitatively expressed in terms of the mean channel number in the fluorescence histogram.

No difference was observed between apoE3 and apoE4 in terms of their effects on the amounts of internalized Aß, which was estimated from the Aß concentration remaining in the medium. However, while the intracellular concentration of Aß gradually declined after reaching peak in the presence of apoE3, there was no such decline in the case of apoE4. Consistent results were also obtained by fluorescence microscopy. These findings may mean that apoE3 acts so as to enhance the clearance of the Aß that has accumulated in the cell (after forming a complex with this Aß). These observations were confirmed using FITC-labeled apoE isoforms as the tracers. Interestingly, the intracellular level of apoE2 is lower than that of apoE3 or apoE4, regardless of the existence of Aß. On the other hand, the intracellular level of apoE4 was similar to that of apoE3 in the absence of Aß, however, the existence of Aß induced marked accumulation of intracellular apoE4 but not apoE3. This shows that apoE4-Aß complex is hard to be catabolized, compared to apoE4 alone.

ApoE4 exhibits by itself greater neurotoxicity than apoE3 [31]. Therefore, the higher accumulation of Aß in the presence of apoE4 would result in decreased cell viability, induced by neurotoxicity of apoE4. In contrast, Jordan et al [25] suggested that apoE3, but not apoE4, is able to prevent Aß from inducing cytotoxicity as a result of uptake via apoE receptors and enhanced clearance of Aß due to the formation of an apoE3-Aß complex. We also observed that incubation of apoE isoforms with Aß1–42 produced a complex with an apparent molecular weight of 40 kDa that reacted with both anti-apoE and anti-Aß antibodies (data not shown). The complex is regarded as an apoE-Aß complex.

Further, Beffert et al [26] reported that while apoE4 promotes the accumulation of Aß, apoE3 reduces Aß-induced cytotoxicity by enhancing the clearance of toxic extracellular Aß more effectively than apoE4. Our data on apoE3 and apoE4 are consistent with these findings. We also found that the effect of apoE2 on the movement of Aß was quite different from the effects produced by apoE3 and apoE4. Our fluorescence intensity data for intracellular Aß indicate that Aß was hardly taken up into the cells. The low percent reduction in the amount of Aß in the medium containing apoE2 supports this interpretation.

ApoE isoforms are known to influence the development of AD [3,7,8,17,18]. The incidence of AD is significantly lower in subjects who have apoE2 or apoE3, especially apoE2, than those who have apoE4. Each apoE isoform has its own binding affinity to Aß [3,19,21], the rank order being E2>E3>E4 [23]; this may be one reason for the isoform-specific nature of AD development.

In this study, the amounts of induced apoE-Aß complexes probably differed among the participating apoE isoforms. Nevertheless, the promotion of a higher avidity binding of Aß to the cell surface and the weak inhibition of the internalization of Aß, which were the observed effects of apoE2, could be the reason for the lower incidence of AD in subjects who have apoE2. ApoE3 is associated with a lower incidence of AD than apoE4. The explanation for this may be quite different from that proposed for the influence of apoE2: namely, that apoE3 facilitates the clearance of internalized Aß. In contrast, apoE2 may be protective against Aß-induced cytotoxicity because it inhibits (or does not facilitate) Aß internalization (because it forms more abundant stable complexes than apoE3 or apoE4).

In medium containing Aß alone, intracellular Aß remained fairly constant; however, Aß concentrations in the medium decreased significantly in a time-dependent manner. These results might be explained as follows: (i) Aß may be taken into cells via the "receptor for advanced glycation end-products" (RAGE) expressed on neurons and microglia [31] and (ii) the Aß added to the medium may bind to apoE3 secreted by the neuroblastoma cells (we confirmed that the neuroblastoma cell line used in this study expressed only apoE3) to form an apoE3-Aß complex, followed by internalization into the cells and rapid clearance from the intracellular space. A time lag in the binding of Aß to secreted apoE3 could be responsible for the constant level of intracellular Aß in medium containing Aß alone.

While free Aß may be taken into neuronal cells via RAGE [32], complexes of Aß with apoE are thought to be taken into cells via receptors recognizing apoE, such as the low density lipoprotein (LDL) receptor [33,34], LDL receptor-related protein [35,36], the very low density lipoprotein (VLDL) receptor [37,38], and the novel lipoprotein receptor [39]. It is known that apoE2 has much lower affinity for LDL receptors than apoE3 and apoE4 [9]. Although additional studies will be required, our data also suggest that the internalization of Aß involves apoE receptors, especially LDL receptors, since it seems likely that the different rates of reduction in Aß in the various media may in part depend on the affinity of a given apoE isoform for the LDL receptor.

In the present study, we used nonlipidated recombinant apoE, because we had found in a previous study [23] that there was no basic difference between lipidated and nonlipidated apoE in the characteristics of the binding of apoE isoforms to Aß1–42. The absolute amount of binding was decreased by lipidation, however, the lipid level in CSF is about 10 mg/L [40], and consequently the effect of lipids on the binding between apoE and Aß1–42 (probably the various receptors too) would presumably be negligible in the CSF.

In conclusion, both apoE3 and apoE4 facilitate the internalization of Aß in the medium, however, only apoE3 promotes the clearance of internalized Aß. In contrast, apoE2 has no effect on Aß clearance. These isoform-specific effects of apoE on the interaction between neuroblastoma cells and Aß may be implicated in the physiological mechanism that underlies the participation of apoE in the development of AD.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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