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Annals of Clinical & Laboratory Science 33:68-78 (2003)
© 2003 Association of Clinical Scientists

Internalization of ß-Amyloid Causes Downregulation of Apolipoprotein E mRNA Expression in Neuroblastoma Cells

Kazuyoshi Yamauchi, Minoru Tozuka, Eiko Hidaka, Ichiro Ueno, Kazuyuki Matsuda and Tsutomu Katsuyama
Department of Laboratory Medicine, Shinshu University Hospital and Medical School, Matsumoto, Japan

Address correspondence to Kazuyoshi Yamauchi, Ph.D., Department of Laboratory Medicine, 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
 
Apolipoprotein (apo) E, like ß-amyloid (Aß), is a key component of the senile plaques that characterize Alzheimer’s disease (AD). Understanding how apoE participates in the formation of senile plaques is necessary to clarify the pathogenesis of AD; however, the mechanism remains unknown. In this study, we investigated the changes of cellular apoE and its mRNA level induced by addition of extracellular Aß to neuroblastoma cells. The presence of >=1.0 µmol/L of Aß induced a decrease of apoE mRNA expression and an increase in the immunofluorescence reactivity for intracellular apoE. Both Aß and apoE were observed by electron-microscopy to be localized within lysosomes. The levels of intracellular apoE and its mRNA returned to the steady state time-dependently. These changes were attenuated by treatments with heparinase I or receptor-associated protein. These findings suggest that the internalized Aß, along with cellular apoE, induces downregulation of apoE mRNA via a pathway possibly mediated by apoE receptors and heparin sulfate proteoglycans. A disorder of this physiological response could be linked to the development of AD.

(received 21 May 2002; accepted 16 July 2002)

Keywords: Alzheimer’s disease, apoE, LRP, RT-PCR, electron microscopy, neuroblastoma cells

Abbreviations: Aß, ß-amyloid; apo, apolipoprotein; AD, Alzheimer’s disease; CNS, central nervous system; LDL, low density lipoprotein; VLDL, very low density lipoprotein; LRP, LDL receptor-related protein; APP, amyloid precursor protein; RAP, receptor-associated protein; RT, reverse transcription; PCR, polymerase chain reaction; FITC, fluorescein isothiocyanate; FCS, fetal calf serum; DMSO, dimethylsulfoxide; DEPC, diethylpyrocarbonate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAB, diaminobenzidine


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Alzheimer’s disease (AD), a cause of senile dementia, is characterized by presence of extracellular neuritic plaques within brain tissue [1–3]. The primary component of this hallmark structure is ß-amyloid (Aß), a 39–43 residue peptide generated by a splicing of ß-amyloid precursor protein [4,5]. Previous studies demonstrated that apolipoprotein (apo) E accumulates as a complex with Aß in this amyloid deposit [1,3,6,7], so apoE is a possible causative factor for late-onset and sporadic AD.

ApoE, one of the main apolipoproteins in the central nervous system (CNS) [8–10], is produced predominantly by astrocytes and microglia, and is involved in the transportation of cholesterol and phospholipids within the CNS [11,12]. In addition, the apoE expressed in the CNS may contribute to growth and repair of the nervous system [9,10]. Further, it has been suggested that members of the low density lipoprotein (LDL)-receptor family, such as LDL receptor [10,13], LDL receptor-related protein (LRP) [14,15], and very low density lipoprotein (VLDL) receptor [16,17] may be involved in the metabolism of apoE-containing lipoproteins within the CNS, since these receptors are expressed in neural cells. In particular, LRP, which participates in the metabolism of remnant lipoproteins in the liver [18], has been proposed as a possible participant in the development of AD on the grounds that it accumulates in neuritic plaques together with Aß and apoE [15]. It has been suggested that LRP may play a role in the metabolism of amyloid precursor protein (APP) by mediating endocytosis of a secreted form of APP [19]. However, the biological functions of apoE and its receptors within the CNS, and the mechanism by which they affect the progression of AD, have yet to be described in detail.

There is no doubt that formation of the apoE-Aß complex is the cause of the deposition of neuritic plaques, while the formation of this complex appears to be an essential physiological response representing the initial step in the catabolism of extracellular Aß. If this process or the subsequent signal-transduction pathway becomes disordered, extracellular Aß deposition may be promoted and the progression of AD facilitated. Hence, the interaction between apoE and Aß could be a critical trigger-point in the development of AD, and a knowledge of how apoE behaves towards extracellular Aß is of importance if we are to clarify fully the pathogenesis of AD.

In the current study, we set out to evaluate the behavior of cellular apoE towards extracellular Aß. To this end, we analyzed changes occurring in levels of cellular apoE and its mRNA in neuroblastoma cells cultured in exogenous Aß-containing medium. We used flow-cytometric analysis, real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR), and electron microscopy. In addition, we performed experiments with heparinase I or receptor-associated protein (RAP) to investigate the mechanisms by which extracellular Aß affects cellular apoE and the expression of apoE mRNA in neuroblastoma cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials.  Aß1–42 peptide was purchased from Alexis Co. (San Diego, CA). Anti-apoE antibody (rabbit), anti-Aß antibody (rabbit), fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (goat), and horseradish-peroxidase (HRP)-conjugated anti-rabbit IgG (goat) were from Dako (Glostrup, Denmark), IBL Co. (Gunma, Japan). Immunotech (Marseilles, France), and MBL Co. (Nagoya, Japan), respectively. Heparinase I was from Sigma Co. (St. Louis, MO), RAP and anti-LRP antibody (mouse) from Progen Biotechnik (GMBH, Heidelberg, Germany), and anti-LDL receptor (mouse) from Oncogene Research Products (Cambridge, MA).

Cell line.  OAN cells [20], a gift from Dr. Garrett M. Brodeur, were maintained in RPMI medium 1640 (Life Technologies) plus 1% Antibiotic Antimyotic (Sigma) containing fetal calf serum (FCS) (Life Technologies) in 5% CO2-air at 37°C.

Preparation of conditioned media.  Aß1–42 peptide (500 µg) was dissolved in 20 µl of dimethyl sulfoxide (DMSO), to which was added 980 µl of sterilized phosphate-buffered saline (PBS). After the solution had been filtered (0.22 µm membrane), it was added to serum-free RPMI Medium 1640 containing 1% Antibiotic Antimyotic to give final concentrations of 0.1, 1.0, or 10.0 µM for Aß1–42. We also prepared heparinase I-containing medium, RAP-containing medium, heparinase I plus Aß1–42-containing medium, and RAP plus Aß1–42-containing medium. The final concentrations of heparinase I and RAP were 8.0 u/ml and 1.0 µmol/L, respectively.

Cell culture.  Cells (1 x 104/ml) were plated in RPMI medium 1640 plus 1% Antibiotic Antimycotic containing 10% FCS, and grown on 20 mm dishes at 37°C in 5% CO2-humidified atmosphere. After the cells had grown to confluency, the medium was aspirated, and the cells adhering to the bottom of the dish were washed with PBS. The prepared conditioned media (see above) were added, and the cultures were allowed to proceed under conditions described above.

Extraction of total cellular RNA, and reverse transcription reaction.  Total RNA was isolated from OAN cells by the acid guanidine thiocyanate-chloroform extraction method [21] using Isogen (Nippon Gene, Tokyo, Japan). The extracted RNA was added to a solution containing 600 µl of isopropylalcohol and 1 µl of glycogen, and stored at -80°C until use.

The extracted RNA was washed with 80% ethanol solution, and resuspended in diethylpyro-carbonate (DEPC)-treated water containing 20 U of RNase inhibitor (Promega Corp., Madison, WI). It was denatured for 5 min at 80°C, and then rapidly chilled on ice. An aliquot containing 1 µg of total RNA was used for the RT reaction, which was carried out at 42°C for 60 min using Moloney murine leukemia-virus reverse transcriptase (Life Technologies) and oligo dT primer (Promega).

Real-time quantitative RT-PCR.  The quantification of apoE mRNA levels in neuroblastoma cells was performed using an ABI Prism 7700 Sequence-Detection System (Perkin-Elmer Applied Bio-systems, Foster City, CA), the basis of which is the continuous optical monitoring of the progress of a fluorogenic PCR [22]. Specific primers and a TaqMan Probe for apoE were designed with the aid of the Primer Express program (Perkin-Elmer Applied Biosystems). Forward and reverse primers for apoE were 5'-TGCAGACACTGTCTGAGCAGGT-3' and 5'-GGAACTGGAGGAACAACTGACC-3', respectively. The forward and reverse primers were hybridized to exons 3 and 4 of the human apoE gene, respectively, and thus did not amplify human genomic DNA. The TaqMan probe was 5'-6-carboxyfluorescein (FAM)-CGAGACCATGAAGGAGTTGA AGGCCTACA-6-carboxy-N, N, N', N'-tetramethylrhodamine (TAMURA)-3', synthesized by PE Applied Biosystems.

A standard curve representing 10-fold serial dilutions of a partial apoE cDNA, which was subcloned into pCR2.1 (Invitrogen, San Diego, CA), was used for linear regression analysis. Reaction mixtures for PCR (50 µl) were prepared by mixing 5 µl of synthesized cDNA solution with 2x TaqMan Universal PCR Master Mix (Perkin-Elmer Applied Biosystems), 500 nM of each primer, and 250 nM of the TaqMan probe. These prepared samples were placed in the analyzer, and PCR was carried out at 50°C for 2 min, 95°C for 10 min, followed by 50 cycles of 95°C for 15 sec, and 60°C for 60 sec.

To normalize the apoE mRNA expression level, a housekeeping gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] was also quantified in the same reactions using TaqMan GAPDH control reagents (Perkin-Elmer Applied Biosystems). The expression level of apoE mRNA was reported as the ratio of apoE mRNA to GAPDH mRNA. Each assay was performed in triplicate.

Fluorescence microscopic analysis.  Cultured cells on glass coverslips were fixed in 20% formalin neutralized buffer solution (Wako Pure Chemicals, Osaka, Japan). To analyze intracellular apoE, the cells were permeabilized using 0.5% saponin and 0.5% bovine serum albumin (BSA) in PBS containing 0.1% NaN3 (permeabilizing solution) for 10 min at room temperature. The non-specific binding sites were blocked by incubation with 10-times volume of PBS containing 10% goat serum (blocking buffer). For the analysis of the cell-surface LDL receptor or LRP, cells were not permeabilized. To these cells, we added 100 µl of a 100-fold dilution of specific antibodies in PBS containing 1% BSA, and incubated for 60 min at room temperature. After three washes with PBS, we added 100 µl of a 100-fold dilution of FITC-conjugated anti-rabbit or mouse IgG, and allowed incubation to proceed for 30 min at room temperature. The coverslips were washed with PBS 3 times and the cells mounted in glycerin for fluorescence microscopic study.

Flow-cytometric analysis of cellular apoE.  Flow-cytometry was carried out as described previously [23]. Briefly, cells, suspended in 200 µl of PBS, were fixed with 20% formalin neutralized buffer solution (Wako Pure Chemicals), followed by permeabilization with the aforementioned permeabilizing solution for 10 min at room temperature. The non-specific binding sites were blocked by incubation with 10-times volume of blocking buffer. We then added 100 µl of a 50-fold dilution of anti-apoE antibody in PBS containing 1% BSA. After a 60-min incubation in iced water followed by 3 washes with PBS, we added 100 µl of a 100-times dilution of FITC-conjugated anti-rabbit IgG, and incubation in iced water was allowed to proceed for 60 min. After 3 washes with PBS, the cells were stored in iced water prior to analysis within 1 hr by FACScan (Becton Dickinson, Franklin Lakes, NJ).

Electron microscopy.  Neuroblastoma cells, cultured on a Lab-Tek Chamber Slide (Nalge Nunc, Copenhagen, Denmark), were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) (PB) for 3 hr at 4°C. They were then washed with cooled PBS, permeabilized using 0.01 % saponin in 4 % paraformaldehyde for 2 min at 4°C, and fixed with 4% paraformaldehyde overnight at 4°C. After washing with chilled PBS, immunostaining with 20-fold anti-apoE or 2-fold anti-Aß was carried out as previously described [24]. After reaction with HRP-anti-rabbit IgG, the cells were re-fixed with 1% glutaraldehyde in PB.

Non-specific peroxidase activity was inhibited by incubation with diaminobenzidine (DAB) solution (without H2O2), containing 0.01 M NaN3 and 1% DMSO, and development was allowed to proceed with DAB solution containing H2O2. The cells were fixed with 2% osmium tetroxide in PB, dehydrated through a graded ethanol series, and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate, and observed using a JEOL JEM1010 transmission electron microscope (JEOL, Tokyo, Japan) at 60 kV accelerating voltage. Negative controls were prepared by omitting the primary antibody.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of Aß on apoE mRNA expression in neuro-blastoma cells.  To assess the effect of exogenous Aß on apoE mRNA expression in neuroblastoma cells, we quantified the apoE mRNA in cells cultured in conditioned media containing Aß1–42 peptide (using the quantitative real-time PCR system). The expression levels of apoE mRNA were normalized with respect to that of GAPDH mRNA, and reported as the apoE:GAPDH mRNA ratio [mRNA (apoE/GAPDH)]. This ratio in control cells averaged 0.443 ± 0.043 (mean ± SD, n = 12). In cells cultured in 0.1, 1.0 or 10.0 µmol/L Aß-containing medium, the ratio showed respectively almost no change, 20% suppression (p <0.01) and 60% suppression (p <0.001) (Fig. 1Go). The expression level of GAPDH mRNA was unaffected by the presence of Aß.



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  		Fig. 1 . Effect of Aß on the expression level of apoE mRNA in neuroblastoma cells. The prepared Aß1–42 solutions were added to serum-free RPMI medium 1640 to obtain a final Aß concentration of 0 (control medium), 0.1, 1.0, or 10 µmol/L. Neuroblastoma cells were cultured in these conditioned media. After 1 hr, the expression level of apoE mRNA was analyzed. Each value (mean ± SD from triplicate measurements in each of 3 separate experiments) represents the apoE:GAPDH mRNA ratio (*p <0.01, **p <0.001).

 
Effect of Aß on cellular apoE.  To assess Aß-induced change of cellular apoE, we analyzed the immunoreactivity of intracellular apoE by immunofluorescence staining followed by flow-cytometric analysis. As shown in Fig. 2aGo, in contrast to the situation seen with the expression level of apoE mRNA, immunofluorescence intensity for cellular apoE was strengthened after culture for 1 hr in medium containing 1.0 µmol/L Aß.



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  		Fig. 2. Immunofluorescence staining and flow-cytometric analysis for intracellular apoE. Neuroblastoma cells were cultured in serum-free RPMI medium 1640 either containing (+Aß) or not containing (control) 1.0 µM Aß1–42. After 1 hr, intracellular apoE was analyzed by immunofluorescence staining (panel a) and flow cytometry (panel b) using anti-apoE antibody. The vertical and horizontal axes show the number of counted cells and the fluorescence intensity, respectively. The scale bar in panel a represents 20 µm.

 
Fig. 2bGo shows histograms of fluorescence intensities obtained from neuroblastoma cells cultured in the presence or absence of 1.0 µmol/L Aß. The mean channel of fluorescence intensity for cellular apoE in cells not treated with Aß was 28.92 ± 2.47. When cells were cultured in Aß-containing medium, the peak was shifted to the right, the mean channel of fluorescence intensities being raised to 51.55 ± 10.04.

Time-related changes in cellular apoE and expression of apoE mRNA.  To investigate the change in cellular apoE induced by exogenous Aß in more detail, we examined the time-related changes in both the immunoreactivity for cellular apoE and the expression level of its mRNA. As shown in Fig. 3Go, the apoE mRNA expression shown by cells in 1.0 µmol/L Aß-containing medium was decreased by about one-half (compared to control cells) after 3 hr in culture. In such cells, the immunofluorescence intensity for cellular apoE was significantly increased, by about 2-fold compared to that of control cells, the peak value being reached after 1 hr in culture. The level of cellular apoE and that of its mRNA both returned to the steady state after 8 hr in culture.



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  		Fig. 3. Time-related changes effects of Aß on cellular apoE and expression level of apoE mRNA. Prepared Aß1–42 solutions were added to serum-free RPMI medium 1640 to give a final Aß concentration of 1.0 µmol/L. Neuroblastoma cells were cultured in this conditioned medium for 1, 3, 5, 8, or 18 hr, and then analyzed for intracellular apoE (closed circle) and the expression level of apoE mRNA (open circle) by flow cytometry (using anti-apoE antibody) and real-time quantitative RT-PCR, respectively. The values are mean ± SD, derived from triplicate determinations in each of 3 separate experiments. Immunointensity for cellular apoE is expressed relative to baseline value (0 hr). The expression level of apoE mRNA is represented by the apoE:GAPDH mRNA ratio.

 
Electron microscopic analysis.  To assess the effect of exogenous Aß on the movement of cellular apoE, ultrastructural immunocytochemistry of the apoE present in neuroblastoma cells was performed by the pre-embedding staining method for electron microscopy. As shown in Fig. 4Go, after a 1-hr culture in 1.0 µmol/L Aß-containing medium, apoE was clearly localized to single-membrane-bound bodies with a doughnut-like structure, the characteristics of lysosomes [25]. The localization of Aß was similar to that of cellular apoE. Both immunointensity levels peaked after 1 hr in culture, and were substantially diminished after 3-hr culture (data not shown). Cells incubated with PBS containing 1% BSA instead of specific primary antibody gave negative results.



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  		Fig. 4. Ultrastractural immunocytochemical determination of the localization of apoE and Aß. Neuroblastoma cells were cultured in serum-free RPMI medium 1640 either containing (+ Aß) or not containing (-Aß) 1.0 µmol/L Aß1–42. After 1 hr, the localization of apoE and that of Aß was analyzed with the pre-embedding staining method for electron microscopy. Cells incubated with PBS containing 1% BSA instead of anti-apoE or anti-Aß antibody gave negative results (control). Scale bar, 2 µm.

 
Effect of heparinase and RAP treatment.  To investigate the mechanisms by which exogenous Aß may affect cellular apoE and the expression of apoE mRNA in neuroblastoma cells, we treated cells with heparinase I or RAP for 2 hr at 37°C, followed in each case by a 1-hr incubation with or without 1.0 µmol/L Aß. The Aß-induced increase in immunofluorescence intensity for intracellular apoE and the Aß-induced suppression of the expression of apoE mRNA were each attenuated by heparinase I treatment (0–15 u/ml) in a dose-dependent manner (data not shown). At 8.0 u/ml of heparinase I (a) the Aß-induced increase in the immunofluorescence intensity for cellular apoE was suppressed to 67% of the value obtained in Aß-incubated cells not treated with heparinase (p <0.05), while (b) the expression level of apoE mRNA (which was reduced by Aß) was restored to 82% of the level seen in control cells (p <0.01, Fig. 5aGo).




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  		Fig. 5. Effects of heparinase and RAP treatment on cellular apoE and the expression level of its mRNA. Neuroblastoma cells were incubated for 2 hr in serum-free RPMI 1640 medium containing (+) or not containing (-) 8.0 u/ml of heparinase I (panel a) or 1.0 µmol/l of RAP (panel b), followed by 1 hr culture in serum-free RPMI 1640 medium containing (+) or not containing (-) 1.0 µmol/L Aß. The intracellular apoE level and the expression level of apoE mRNA in these cells were analyzed by flow cytometry (using anti-apoE antibody) and real-time quantitative RT-PCR, respectively. The values shown are the means ± SD from triplicate determinations in each of 3 separate experiments, and are expressed relative to those obtained for control cells, cultured in serum-free medium alone (*p <0.05, **p < 0.01).

 
We also investigated the effect of the apoE-receptor antagonist RAP. Although we found no evidence of dose-dependency (data not shown), the Aß-induced changes were substantially attenuated by RAP treatment as they had been by heparinase treatment. The Aß-induced increase in the immunofluorescence intensity for cellular apoE was 59% of that seen in Aß-incubated cells not treated with RAP (p<0.05), while apoE mRNA expression was restored by RAP to 90% of the level in control cells (p <0.05, Fig. 5bGo). Neither heparinase I nor RAP by themselves had any effect on cellular apoE (Fig. 5a,bGo).

Fluorescence microscopic analysis of LDL receptor and LRP.  To investigate the effect of Aß on the expression of LDL receptor and LRP on the surface of neuroblastoma cells, immunofluorescence staining with specific antibodies was performed using cells cultured in 1.0 µmol/L Aß-containing medium (Fig. 6Go). Immunofluorescence intensity for the LDL receptor on the cell surface was no different regardless of the presence or absence of Aß in the medium. In contrast, immunofluorescence intensity for LRP was significantly strengthened in the presence of 1.0 µmol/L Aß. The intensity then declined time-dependently, and returned to the steady state at about 3 hr (data not shown). Cells incubated with PBS containing 1% BSA instead of the specific primary antibodies gave negative results (data not shown).



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  		Fig. 6. Immunofluorescence staining of neuroblastoma cells cultured in Aß-containing medium. Neuroblastoma cells on glass coverslips were cultured in serum-free RPMI medium 1640 containing (+Aß) or not containing (-Aß) 1.0 µmol/L Aß1–42. After 1 hr in culture, they were fixed, and then immunostained for LDL receptor or LRP using specific antibodies. Scale bar, 20 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Evidence suggests that apoE is closely implicated in the development of AD. Bales et al [26] and Holtzman et al [27,28], using a mouse model of AD, showed that apoE promotes deposition and fibrillarization of Aß. These findings strongly suggest that interaction between apoE and Aß contributes to Aß deposition and neuritic-plaque formation. In addition, interactions before Aß deposition may affect Aß clearance from the extracellular space (see below). A precise understanding of the significance of the interaction of apoE with Aß, in particular the process resulting in formation of neuritic plaques, is important if we are to clarify the role played by apoE in the pathogenesis of AD. In the current study, we used neuroblastoma cells to investigate the changes of apoE mRNA expression and cellular apoE induced by exogenous Aß.

The expression level of apoE mRNA was decreased by incubation with >=1.0 µmol/L of Aß. This was not due to a toxic effect of Aß, since the expression level of GAPDH mRNA was roughly constant before and after incubation with Aß. The suppression of mRNA would be expected to cause a decrease in the coded protein itself; however, the amount of cellular apoE actually showed a significant increase. This Aß-induced increase in the amount of cellular apoE is consistent with previous findings reported by LaDu et al [29], although unlike us they found no change in the steady-state level of apoE mRNA following Aß-stimulation. We believe our result may be quantitatively more accurate than theirs, since we evaluated the expression level of the mRNA using a highly sensitive and quantitative real-time RT-PCR [22]. In our experiment, we could detect no change in the levels of cellular apoE or its mRNA when we used 0.1 µmol/L Aß. Possibly, in the brain a local increase in Aß concentration above some threshold level may lead to marked decrease in apoE mRNA, whereas within the physiological range of Aß levels the response may be too small to be detectable. It is well known that apoE binds to Aß in isoform-specific manner, and forms complexes with it [30–32]. In addition, studies have suggested that the apoE-Aß complex is taken up into cells and cleared from the extracellular space [31,33,34].

Taking all this together, we suspect that the contrary behavior of apoE and its mRNA described above is due to downregulation of apoE mRNA being induced by a facilitated internalization of apoE within the cell accompanied by enhanced clearance of extracellular Aß. The changes in cellular apoE and its mRNA were roughly synchronous, with intracellular apoE possibly peaking somewhat before the maximum decrease in apoE mRNA expression. Although we are unable to infer whether or not an apoE-Aß complex was formed in our experiments, we did establish that apoE and Aß were both localized within lysosomes (by immunoelectron microscopic analysis).

This finding eliminates the possibility that the increase in the immunointensity for cellular apoE was the result of an interference with the secretion of apoE due to a toxic effect of Aß, and supports the idea that exogenous Aß is internalized and then catabolized with cellular apoE, as demonstrated previously [31,33,34]. In addition, this finding leads to the following notions: (a) that most Aß is taken up into cells via apoE receptors, and (b) that a signal-transduction pathway mediated by apoE receptors is linked to the downregulation of apoE mRNA expression.

In the current study, the Aß-induced effects (increase in immunoreactivity for intracellular apoE and suppression of apoE mRNA expression) were significantly attenuated by pretreatment of cells with either heparinase I, which removes the sulfated glycosaminoglycan side-chains from proteoglycans, or 1.0 µmol/L RAP, an antagonist for apoE receptors. These findings appear to indicate that extracellular Aß is mainly taken up into cells via a pathway linked to apoE receptors and HSPGs (heparin sulfate proteoglycans), and cleared from the extracellular space [18,19,35,36]. It seems likely that the interaction of extracellular Aß, or the apoE-Aß complex, with neuronal cells via this apoE receptors-HSPGs pathway is a starting point (trigger) for the subsequent downregulation of apoE mRNA.

Various apoE receptors have been suggested to be implicated in catabolism of Aß [16,17,37,38]. LRP has been highlighted in AD pathology, because LRP immunoreactivity is abundant on neuronal cell bodies, and because it is a major neuronal receptor for apoE [39,40]. It has been suggested that LRP may mediate the clearance of Aß or the apoE-Aß complex from the extracellular space [35,41]. In addition, the neurite-extension effect of apoE3, but not that of apoE4, is blocked by RAP and anti-LRP antibody [42], as well as by heparinase treatment [43]. Jordan et al [31] suggested that apoE3, but not apoE4, protects rat hippocampal neurons against Aß-induced neurotoxicity, a process inhibited by RAP. These findings suggest that the LRP-HSPGs pathway may play a critical role in the metabolism of apoE-containing lipoproteins, and may be closely implicated in AD pathology.

LaDu et al [29] recently concluded that the LDL receptor, but not LRP, is involved in mediating the Aß-induced changes in apoE (on the basis of the difference between LRP and LDL receptor in binding affinity towards RAP). We observed that RAP, at concentrations less than 250 nmol/L (the dissociation constant of RAP for the LDL receptor [44]), did not block the Aß-stimulated increase in the immunoreactivity for intracellular apoE or the suppression of apoE mRNA expression (data not shown).

We cannot deduce which receptor might take part in the internalization of Aß and the down-regulation of apoE mRNA solely on the basis of experiments using RAP, because a low concentration RAP would be endocytosed too rapidly to allow us to examine its precise effect. Nevertheless, the results of our immunostaining for apoE receptors support the notion that LRP, not the LDL receptor, may be linked to internalization of Aß and the consequent downregulation of apoE mRNA expression.

It is well known that polymorphism of apoE has relevance for development of AD. In particular, apoE4 is believed to be a risk factor for sporadic and late-onset familial AD on the grounds that the incidence of AD is significantly higher in subjects who possess apoE4 [7,45,46]. The neuroblastoma cells used in the current study expressed apoE3, and if the binding of apoE to Aß and the subsequent internalization of the apoE-Aß complex really is apoE isoform-specific, as demonstrated by several previous studies [30–32], it would be interesting to examine how apoE4- or apoE2-expressing cells might behave when confronted with excessive Aß. That, too, is of importance for AD pathology.

In conclusion, we suggest that the expression level of apoE mRNA in neuroblastoma cells is downregulated when excessive extracellular Aß is internalized into the cell to join cellular apoE, and thus cleared from the extracellular space, and that apoE receptors are closely implicated in this process. We hypothesize that: (a) in the normal brain, the changes in cellular apoE and the expression level of apoE mRNA may represent a physiological response against the excessive extracellular Aß, and (b) if this process is incompetent or lost, the accumulation of extracellular Aß may be facilitated, leading to the development of neuritic plaques and possibly contributing to the pathogenesis of AD.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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