Interaction of Apolipoprotein J-Amyloid beta -Peptide Complex with Low Density Lipoprotein Receptor-related Protein-2/Megalin
A MECHANISM TO PREVENT PATHOLOGICAL ACCUMULATION OF AMYLOID beta -PEPTIDE*

(Received for publication, May 15, 1997)

Samar M. Hammad , Sripriya Ranganathan , Elena Loukinova Dagger , Waleed O. Twal and W. Scott Argraves §

From the Cell Biology and Anatomy Department, Medical University of South Carolina, Charleston, South Carolina 29425-2204

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Apolipoprotein J (apoJ) has been shown to be the predominant amyloid beta -peptide (Abeta )-binding protein in cerebrospinal fluid. We have previously demonstrated that the endocytic receptor low density lipoprotein receptor-related protein-2/megalin (LRP-2), which is expressed by choroid plexus epithelium and ependymal cells lining the brain ventricles and neural tube, binds and mediates cellular uptake of apoJ (Kounnas, M. Z., Loukinova, E. B., Stefansson, S., Harmony, J. A., Brewer, B., Strickland, D. K., and Argraves, W. S. (1995) J. Biol. Chem. 270, 13070-13075). In the present study, we evaluated the ability of apoJ to mediate binding of Abeta 1-40-apoJ complex to LRP-2 in vitro. Immunoblot analysis showed that incubation of apoJ with Abeta 1-40 resulted in the formation of Abeta 1-40-apoJ complex and the inhibition of the formation of Abeta 1-40 aggregates. Using an enzyme-linked immunosorbent assay, an estimated dissociation constant (Kd) of 4.8 nM was derived for the interaction between Abeta 1-40 and apoJ. Enzyme-linked immunosorbent assay was also used to study the interaction of the Abeta 1-40-apoJ complex with LRP-2. The results showed that Abeta alone did not bind directly to LRP-2; however, when Abeta 1-40 was combined with apoJ to form a complex, binding to LRP-2 took place. The binding interaction could be blocked by inclusion of the receptor-associated protein, an antagonist of apoJ binding to LRP-2. When LRP-2-expressing cells were given 125I-Abeta 1-40, cellular uptake of the radiolabeled peptide was promoted by co-incubation with apoJ. When the cells were provided purified 125I-Abeta 1-40-apoJ complex, the complex was internalized and degraded, and both processes were inhibited with polyclonal LRP-2 antibodies. Furthermore, chloroquine treatment inhibited the cellular degradation of the complex. The data indicate that apoJ facilitates Abeta 1-40 binding to LRP-2 and that the receptor mediates cellular clearance of Abeta 1-40-apoJ complex leading to lysosomal degradation of Abeta 1-40. The findings support the possibility that LRP-2 can act in vivo to mediate clearance of the complex from biological fluids such as cerebrospinal fluid and thereby play a role in the regulation of Abeta accumulation.


INTRODUCTION

A hallmark feature of Alzheimer's disease is the accelerated cerebral accumulation of amyloid beta -protein (Abeta ),1 a small 39-42-residue proteolytically derived fragment of amyloid beta -precursor protein (1, 2). The accumulation takes the form of spherical extracellular deposits of Abeta fibrils in the vicinity of morphologically abnormal axons and dendrites. Associated with these so-called plaques are microglia and astrocytes. The mechanisms that lead to accumulation of Abeta are still obscure but represent an area of intense investigation. Whereas much emphasis is currently placed on trying to determine the mechanism(s) of Abeta biosynthesis from amyloid beta -precursor protein processing (3, 4), little is being done on determining possible mechanisms that mediate the catabolism of Abeta . Catabolic processes may prevent the extracellular accumulation of Abeta that is expressed under normal physiological conditions yet does not accumulate to the extent seen in Alzheimer's disease or Down's syndrome.

Abeta can be found in cerebrospinal fluid and blood in complex with apolipoprotein J (apoJ) or apolipoprotein E (apoE) (5, 6). Whereas apoE has been reported to promote Abeta fibrilogenesis (7-9), apoJ has been shown to slow the formation of Abeta aggregates and may therefore act to maintain Abeta in a soluble form and prevent it from forming pathological fibrils (10). Our discoveries that LRP-22 is an endocytic receptor for apoJ (11) and LRP-2 is expressed by cells that are in contact with cerebrospinal fluid (choroid plexus and ependymal cells) (12) prompted us to hypothesize that LRP-2 may mediate clearance of Abeta complexed with apoJ, thereby controlling the accumulation of Abeta . In the present study, we used in vitro solid phase binding assays as well as cellular internalization and degradation assays to evaluate the roles of apoJ and LRP-2 in mediating cellular clearance of Abeta .


EXPERIMENTAL PROCEDURES

Proteins

Human apoJ was purchased from Quidel (San Diego, CA). Synthetic Abeta fragment 1-40 and ovalbumin were obtained from Sigma. Bovine serum albumin was purchased from U. S. Biochemical Corp. Human RAP was expressed as a glutathione S-transferase fusion protein in bacteria and prepared free of glutathione S-transferase as described by Williams et al. (13). LRP-2 was purified from extracts of porcine kidney by affinity chromatography using a column of RAP coupled to Sepharose as described previously (36).

Antibodies

The mouse monoclonal antibody to LRP-2 designated 1H2 was provided by Dr. Robert McCluskey (Massachusetts General Hospital, Boston, MA). Mouse monoclonal antibody to human apoJ (mAb 1D11) was obtained from Dr. Judith Harmony (University of Cincinnati College of Medicine, Cincinnati, OH). Mouse monoclonal antibody to human Abeta (mAb 4G8) was purchased from Senetek (Maryland Heights, MO). Rabbit anti-LRP-2 IgGs (rabbit 6286) were isolated by immunoaffinity chromatography on a column of porcine LRP-2 coupled to CNBr-activated Sepharose (Pharmacia Biotech Inc.) with minor modification to a previously described procedure (11). IgG was sequentially eluted using 100 mM glycine, pH 2.3, followed by 100 mM triethylamine, pH 11.5, and the combined eluates were dialyzed against 50 mM Tris, pH 7.4, 150 mM NaCl. The polyclonal anti-LRP-2 IgG preparation was absorbed on a column of RAP-Sepharose followed by selection on a column of protein G-Sepharose. Control rabbit IgG was isolated from the preimmune serum of rabbit 6286 by protein G-Sepharose chromatography.

Formation of Abeta -ApoJ Complex

ApoJ was combined with synthetic Abeta 1-40 at a 1:15 molar ratio in PBS as described previously (14) and incubated for 24 h at 37 °C. Typically, a 1:15 molar ratio was maintained for preparing complexes of unlabeled Abeta 1-40 and apoJ, although the total protein concentration varied according to the assay. For SDS-PAGE and immunoblotting analyses the concentration of apoJ was 0.95 µM and Abeta 1-40 was 15 µM, whereas for solid phase binding assays, apoJ was 0.095 µM and Abeta 1-40 was 1.5 µM. As a control, ovalbumin was substituted for apoJ.

Preparation and Purification of 125I-Abeta -ApoJ Complex

Abeta 1-40 peptide (300 µg in Dulbecco's PBS (dPBS)) was radioiodinated by the IODO-GEN (Pierce) method using 2 mCi of Na125I (Amersham Life Science, Inc.). Unincorporated [125I]iodine was removed by Sepharose G-15 chromatography using a 0.7 × 18-cm column equilibrated with dPBS. Radioiodinated Abeta 1-40 peptide (100 µg) was combined with apoJ (50 µg in dPBS) at a 30:1 molar ratio and incubated for 36 h at 37 °C. Following the incubation, radiolabeled Abeta -apoJ complex was separated from free 125I-labeled Abeta by gel filtration chromatography using a model 650E Waters protein purification system (Waters, Milford, MA) and a Superdex-200HR column (Pharmacia) equilibrated with dPBS. The integrity of the complex was evaluated by electrophoresis under non-denaturating conditions on 4-12% polyacrylamide, Tris/glycine-containing gels (Novex, San Diego, CA) followed by autoradiography.

Enzyme-linked Immunosorbent Assays (ELISA)

Microtiter wells were coated with LRP-2, Abeta , or BSA (each at 3 µg/ml) in 150 mM NaCl, 50 mM Tris, pH 8.0 (TBS) containing 5 mM CaCl2 for 18 h at 4 °C. Unoccupied sites were either blocked with TBS containing 3% nonfat milk or treated with PBS containing 0.1% N-octyl-beta -D-glucopyranoside (OG) (Calbiochem). For those wells that were blocked with TBS containing 3% nonfat milk, all subsequent incubations were carried out in TBS containing 3% nonfat milk plus 0.1% OG. For wells treated with PBS containing 0.1% OG, all subsequent incubations were carried out in PBS containing 0.1% OG. Bound proteins were detected using mouse monoclonal antibodies, sheep anti-mouse IgG-horseradish peroxidase (Amersham International, Buckinghamshire, UK), and the chromogenic substrate o-phenylenediamine (Sigma) (1 mg/ml in 12 mM citric acid monohydrate, 25 mM dibasic sodium phosphate, pH 5.0, 0.0014% hydrogen peroxide). Binding data from ELISA were analyzed using a form of the binding isotherm as described by Ashcom et al. (15).

Immunoblotting

To analyze Abeta -apoJ complexes by SDS-PAGE, samples were electrophoresed on 10-20% acrylamide gradient, Tricine-containing gels (Novex) in the presence of SDS. The separated proteins were electrophoretically transferred to ProtranTM nitrocellulose membranes (Schleicher & Schuell) in Tris/glycine-methanol buffer for 2 h at 70 V. After transfer, the membranes were incubated with 5% nonfat dry milk in TBS (pH 7.4). The membranes were then incubated with monoclonal Abeta or apoJ antibodies followed by sheep anti-mouse IgG-horseradish peroxidase (Amersham) diluted in 5% nonfat milk, TBS, 0.1% Tween 20. To detect bound antibodies, the membranes were incubated with ECLTM Western blotting chemiluminescent reagent (Amersham) and exposed to BiomaxTM MR film (Eastman Kodak Co.).

Assay of Cellular Internalization and Degradation of 125I-Abeta and 125I-Abeta -ApoJ Complex

To evaluate the effect of apoJ on the internalization of 125I-Abeta , mouse teratocarcinoma F9 cells (ATCC CRL 1720) were treated for 6 days with retinoic acid (RA) and dibutyryl cyclic AMP (Bt2cAMP) as described previously (30), released by trypsin-EDTA, and reseeded onto gelatin-coated 383-mm2 wells of 12-well plates (1.5 × 105 cells/well) in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.) containing 10% bovine calf serum (Hyclone Laboratories), 20 mM HEPES, pH 7.4, 100 units/ml penicillin, 100 µg/ml streptomycin (Life Technologies, Inc.) and no RA/Bt2cAMP. The cells were cultured for 18 h at 37 °C, 5% CO2 and then washed with serum-free DMEM containing penicillin and streptomycin. 125I-Abeta (70 nM) in DMEM, 1.5% BSA, 1% Nutridoma serum substitute (Boehringer Mannheim) (DMEM/BSA/SS) containing various amounts of apoJ (1-40 nM) was added to the cells and incubated for 5 h at 37 °C, 5% CO2. The amount of radiolabeled Abeta that was internalized by cells was defined as the amount of radioactivity that remained associated with the cell pellets following trypsin/proteinase K/EDTA treatment (11, 30).

To evaluate the role of LRP-2 in the cellular internalization and degradation of 125I-Abeta -apoJ complex, RA/Bt2cAMP-treated mouse teratocarcinoma F9 cells were cultured as described above. 60 min prior to the addition of 125I-Abeta -apoJ complex, the medium was removed and the cells were treated with DMEM/BSA/SS containing either anti-LRP-2 IgG (200 µg/ml), control rabbit IgG (200 µg/ml), or chloroquine (0.1 mM). 125I-Abeta -apoJ complex (10 nM) in DMEM/BSA/SS or in DMEM/BSA/SS containing anti-LRP-2 IgG (200 µg/ml), rabbit IgG (200 µg/ml), or chloroquine (0.1 mM) was then added and incubated with the cells for 5 or 18 h at 37 °C, 5% CO2. The amount of radiolabeled complex that was internalized was measured as described above. Radioactivity released into the conditioned culture medium that was soluble in 10% trichloroacetic acid was taken to represent degraded ligand. Total ligand degradation values were corrected for non-cellular mediated degradation by subtracting the amount of degradation that occurred when the radiolabeled complex was incubated in wells lacking cells.


RESULTS

Amyloid beta -Protein Binds to ApoJ but Not to LRP-2

ELISAs were used to determine whether synthetic Abeta 1-40 peptide was capable of binding directly to purified LRP-2. As shown in Fig. 1A, LRP-2 did not bind to microtiter wells coated with Abeta 1-40. Likewise, Abeta 1-40 (at concentrations up to 125 nM) did not bind to LRP-2-coated wells (Fig. 1B). In parallel assays, Abeta 1-40 was shown to bind to apoJ in a dose-dependent manner, either when Abeta 1-40 was coated onto microtiter wells and apoJ was introduced in solution phase (Fig. 1A) or when apoJ was coated and Abeta 1-40 was introduced in solution phase (Fig. 1B). The data for solution-phase apoJ binding to immobilized Abeta 1-40 (Fig. 1A) were fit using a hyperbolic function (16), and the half-saturating level of binding (estimated Kd) was determined to be 4.8 nM. This value is in good agreement with the value of 2.0 nM reported by Matsubara et al. (6). By contrast, the binding of solution-phase Abeta 1-40 to immobilized apoJ was not saturable (Fig. 1B). Such non-saturable binding can be expected given that Abeta 1-40 has the ability to self-associate (17). The results indicate that Abeta 1-40 does not bind to LRP-2 but does bind with high affinity to apoJ.


Fig. 1. Abeta 1-40 binds with high affinity to apoJ but not to LRP-2. In panel A, microtiter wells coated with Abeta 1-40 (3 µg/ml, plus a milk block) were incubated with varying concentrations of either apoJ (0.18-400 nM, bullet ) or LRP-2 (0.23-500 nM, open circle ) in PBS containing 0.1% N-octyl-beta -D-glucopyranoside. Bound protein was detected by ELISA using monoclonal antibodies to apoJ or LRP-2. In panel B, microtiter wells coated with either apoJ, LRP-2, or BSA (each at 3 µg/ml, no blocking with milk) were incubated with varying concentrations of Abeta 1-40 (1.37-125 nM) in PBS containing 0.1% N-octyl-beta -D-glucopyranoside; bound peptide was detected by ELISA using monoclonal Abeta antibody 4G8. The plotted values are means of duplicate determinations with the range indicated by bars and are representative of three experiments.
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Amyloid beta -Protein-ApoJ Complex Binds to LRP-2

To generate Abeta -apoJ complex, Abeta 1-40 was incubated with apoJ for 24 h at 37 °C, and complex formation was evaluated by SDS-PAGE and immunoblot analysis. As shown in Fig. 2, this incubation resulted in the formation of an approximately 70-kDa band, immunoreactive with monoclonal Abeta antibody (Fig. 2B, lane 4). The anti-Abeta -reactive 70-kDa band displayed a similar electrophoretic mobility to the 70-kDa apoJ (Fig. 1C, compare lanes 4 and 6). Incubation of Abeta with ovalbumin did not produce a band having a similar molecular mass (Fig. 2B, lane 5). Coomassie Blue staining showed that in the lane containing Abeta , which had been incubated alone for 24 h at 37 °C, there was a single band having a mobility corresponding to ~4 kDa (Fig. 2A, lane 3), consistent with Mr of 4392. However, immunoblot analysis of this lane (Fig. 2B, lane 3) using monoclonal Abeta antibody revealed several immunoreactive species having Mr values of ~8000 and ~12,000 and a high molecular mass band that just entered the gel. These species likely correspond to Abeta dimer, trimer, and aggregate, respectively. Although the dimer, trimer, and aggregated species were not detectable by Coomassie Blue staining, they were immunoreactive with Abeta antibody, indicative perhaps of its preference for multimerized peptide versus the monomeric form. Each of these immunoreactive species was also present in the profile of the Abeta incubated with ovalbumin. However, the Abeta aggregate was missing in the lane containing Abeta incubated with apoJ (Fig. 2B, lane 4). The data indicated that incubation of apoJ with Abeta under the conditions that we described resulted in the formation of a complex of Abeta and apoJ that is stable in SDS. The conditions of SDS-PAGE were insufficient to permit resolution of the Abeta -apoJ complex as a discrete species having a Mr ~4000 greater than apoJ. The results also showed that apoJ inhibited the formation of aggregated Abeta while not perturbing the formation of Abeta dimer and trimer.


Fig. 2. SDS-PAGE evaluation of complex formation of Abeta 1-40 and apoJ. Equal volume aliquots of 15 µM Abeta 1-40 (lane 3), 15 µM Abeta 1-40 plus 0.95 µM apoJ (lane 4), 15 µM Abeta 1-40 plus 0.95 µM ovalbumin (lane 5), 0.95 µM apoJ (lane 6), and (0.95 µM ovalbumin (lane 7) were separated by SDS-PAGE. The gel was stained with Coomassie Blue (panel A) or transferred to nitrocellulose, and the membrane was probed with anti-Abeta (panel B) or anti-apoJ IgG (panel C). Protein solutions were incubated for 24 h at 37 °C prior to analysis. Lane 2 in each panel is blank. Molecular size standards are in lane 1 with their molecular mass values (in kDa) indicated on the left. The data shown are representative of five experiments.
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To evaluate the ability of Abeta -apoJ complex to interact with LRP-2, microtiter wells coated with LRP-2 were incubated with mixtures of Abeta and apoJ or Abeta and ovalbumin that had been preincubated for 24 h at 37 °C. As shown in Fig. 3A, Abeta binding to immobilized LRP-2, as detected by Abeta monoclonal antibody, occurred when Abeta was preincubated with apoJ but not with ovalbumin. Experimental controls showed that neither of the mixtures, Abeta and apoJ or Abeta and ovalbumin, bound to wells coated with BSA (Fig. 3B). When monoclonal antibody to apoJ was used to detect apoJ binding to LRP-2, the half-saturating level of binding of apoJ to LRP-2 was not significantly modified by the inclusion of Abeta (Fig. 3C). The results indicate that apoJ mediates binding of Abeta to LRP-2 and that the affinity of the Abeta -apoJ complex for LRP-2 does not appear to be different from that of apoJ alone.


Fig. 3. Abeta 1-40-apoJ complex binds to LRP-2. Solutions of 1.5 µM Abeta 1-40 plus 0.095 µM apoJ, 1.5 µM Abeta 1-40 plus 0.155 µM ovalbumin, or 0.095 µM apoJ plus 0.155 µM ovalbumin were preincubated for 24 h at 37 °C and then incubated in varying concentrations (0.00067-13.34 µg/ml) with microtiter wells coated with LRP-2 (panels A and C) or BSA (panel B). Bound protein was detected by using monoclonal Abeta antibody (panels A and B) or monoclonal apoJ antibody (panel C). The plotted values are means of duplicate determinations with the range indicated by bars. The data shown are representative of four experiments.
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The Antagonist of ApoJ Binding to LRP-2 Blocks Binding of Abeta -ApoJ Complex to LRP-2

RAP has been shown to inhibit the binding of apoJ to LRP-2 (11). As shown in Fig. 4, incubation of Abeta -apoJ complex with RAP completely blocked the binding of the complex to immobilized LRP-2. This, taken together with the above data, indicates that apoJ can function to bridge the interaction of Abeta with LRP-2.


Fig. 4. Abeta 1-40-apoJ complex binding to LRP-2 is inhibitable by RAP, the antagonist of apoJ binding to LRP-2. Solutions of Abeta 1-40 (1.5 µM) plus apoJ (0.095 µM) preincubated for 24 h at 37 °C were combined with either RAP (100 nM) or ovalbumin (100 nM) and incubated with microtiter wells coated with LRP-2 (panel A) or BSA (panel B). Bound protein was detected by using monoclonal Abeta antibody. The plotted values are means of duplicate determinations with the range indicated by bars. The data shown are representative of three experiments.
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Cellular Endocytosis of Abeta Is Facilitated by ApoJ

To determine whether apoJ might facilitate cellular internalization of Abeta , 125I-Abeta was administered to cultured LRP-2-expressing cells in the presence of varying concentrations of apoJ. As shown in Fig. 5, exogenously added apoJ promoted the internalization of 125I-Abeta in a dose-dependent manner.


Fig. 5. ApoJ facilitates internalization of 125I-Abeta 1-40 by LRP-2-expressing cells. RA/Bt2cAMP-differentiated F9 cells were incubated for 5 h at 37 °C, 5% CO2 with 125I-Abeta 1-40 (70 nM) in the presence of various concentrations of apoJ (1-40 nM). Shown are the amounts of 125I-Abeta 1-40 internalized by the cells. Plotted values are means ± S.D. of triplicate values.
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LRP-2 Mediates Cellular Endocytosis and Degradation of Abeta -ApoJ Complex

We next examined the cellular clearance of Abeta -apoJ complex and evaluated the role of LRP-2 in the process. Radioiodinated Abeta was combined with unlabeled apoJ, and the resulting complex was purified by gel filtration chromatography. Fig. 6A shows the chromatographic profiles of the individual components and the complex-containing mixture. Native gel electrophoretic analysis of the complex-containing fraction is shown in the inset of Fig. 6A. The results indicated that the chromatography procedure permitted isolation of the 125I-labeled complex from the bulk of the unincorporated Abeta ; however, free radiolabeled peptide did copurify with the complex.


Fig. 6. Purification of 125I-Abeta 1-40-apoJ complex by gel filtration chromatography and its internalization by LRP-2-expressing cells. Shown in panel A are the Superdex 200-HR chromatographic profiles of apoJ (10 µg), Abeta 1-40 (10 µg), and a mixture of 125I-Abeta 1-40 (100 µg, 8.6 × 106 cpm/µg) plus apoJ (50 µg) that had been preincubated for 36 h at 37 °C. The inset shows an autoradiograph of selected fractions (peaks 1 and 2) from Superdex 200-HR chromatography of 125I-Abeta 1-40 + apoJ after separation by electrophoresis on a non-denaturing 4-12% polyacrylamide gel. Shown in panel B are the levels of either 125I-Abeta 1-40 or 125I-Abeta 1-40-apoJ internalized by RA/Bt2cAMP-differentiated F9 cells. Cultures were incubated with the respective probe at a concentration of 10 nM in the absence of any competitor or in the presence of anti-LRP-2 IgG or control IgG (each at 200 µg/ml) 18 h at 37 °C, 5% CO2. Plotted values are means ± S.D. of triplicate values and are representative of three experiments.
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Equimolar amounts of 125I-Abeta or 125I-Abeta -apoJ were administered to LRP-2-expressing cells, and the level of internalization of each was measured. As shown in Fig. 6B, there was a 2-fold higher level of 125I-Abeta -apoJ complex internalized as compared with 125I-Abeta alone. In separate experiments, the amount of 125I-Abeta internalized was unchanged by the inclusion of a 1000-fold molar excess of unlabeled peptide (data not shown). Complex internalization was also measured in the presence of function blocking LRP-2 antibodies or control IgGs. As shown in Fig. 6B (and Fig. 7A), anti-LRP-2 IgG inhibited 125I-Abeta -apoJ complex internalization by 59% as compared with treatment with control rabbit IgGs. The results indicate that Abeta -apoJ complex is internalized by LRP-2-expressing cells to a greater extent than Abeta alone and that the internalization of the complex can be inhibited by LRP-2 antibodies, implicating LRP-2 in the clearance process.


Fig. 7. LRP-2 antibodies and chloroquine inhibit degradation of 125I-Abeta 1-40-apoJ complex by LRP-2-expressing cells. RA/Bt2cAMP-differentiated F9 cells were incubated with 125I-Abeta 1-40-apoJ complex (10 nM) in the presence of affinity-purified anti-LRP-2 IgG (200 µg/ml), control rabbit IgG (200 µg/ml), or chloroquine (0.1 mM) for 5 h at 37 °C, 5% CO2. Shown are the amounts of 125I-Abeta 1-40-apoJ internalized (A) and degraded (B) by the cells. Plotted values are means ± S.D. of triplicate values and are representative of duplicate experiments.
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We also evaluated whether the internalized complex was lysosomally degraded as is the case for other LRP-2 ligands including apoJ (11). As shown in Fig. 7, administration of 125I-Abeta -apoJ complex to LRP-2-expressing cells resulted in the internalization and degradation of the complex. Degradation was evidenced by the appearance of trichloroacetic acid-soluble radioactivity in the conditioned culture medium that could be blocked by treatment with chloroquine, an inhibitor of lysosomal proteinase activity (Fig. 7B). Both internalization and degradation of the complex were also inhibited with LRP-2-antibodies. Taken together, the findings indicate that LRP-2 mediates endocytosis of Abeta -apoJ complex leading to its degradation in lysosomes.


DISCUSSION

In this study, we document the ability of the Abeta -apoJ complex to bind to the endocytic receptor LRP-2 in both cell-free and cultured cell assays. Although the physiological relevance of this interaction remains to be established, we have previously hypothesized that it is part of a mechanism in which LRP-2-expressing epithelial cells, such as those of the choroid plexus and ependyma, can clear the Abeta -apoJ complex from the cerebrospinal fluid (11). In support of such a hypothesis is the fact that both apoJ and LRP-2 are expressed at high levels in the choroid plexus epithelium as well as ependymal cells that line the ventricles of the brain and neural tube (12, 18, 19) and are therefore in direct contact with cerebrospinal fluid. In addition to LRP-2 having a possible role in clearance of Abeta -apoJ complex from the cerebrospinal fluid, there is evidence that LRP-2 may have a role in uptake of the complex from the blood by cells of the cerebral vascular endothelium (14, 20). Following our previous report on the identification of LRP-2 as the receptor for apoJ, Zlokovic et al. (14) introduced radiolabeled Abeta -apoJ complex into rat brain vasculature and observed that RAP or monoclonal antibody to LRP-2 decreased the brain uptake of the radiolabeled complex. Whereas these findings indirectly implicate LRP-2 as being responsible for the observed vascular clearance, evidence is needed to show that LRP-2 is indeed expressed by brain vascular endothelial cells. Nevertheless, the results presented in the present study provide direct evidence that LRP-2 is a receptor for the Abeta -apoJ complex, thus establishing that it could serve to mediate Abeta -apoJ endocytosis in any LRP-2-expressing tissue.

Our results also indicate that apoJ inhibits the formation of high molecular weight aggregates of Abeta 1-40 (Fig. 2). This activity is consistent with observations made using a sedimentation assay (10) and with the hypothesis that apoJ serves to maintain Abeta in a soluble form, preventing it from forming insoluble amyloid filaments. Such filaments are the hallmark component of the senile plaque found in brain parenchyma and deposited in the cerebrovasculature of patients with Alzheimer's disease (21, 22). In addition, the Abeta -apoJ interaction may have a cytoprotective effect given that the insoluble aggregated form of Abeta has been shown to be toxic to cultured neuronal cells (23). By contrast to the anti-amyloidogenic and cytoprotective actions of apoJ, apoE has been shown to promote the formation of Abeta fibrils (7, 9). Further studies are required to understand the factors that affect the in vivo interaction of Abeta with apoJ or apoE. It is possible that under normal physiological conditions, the interaction between apoJ and Abeta is favored over that of apoE to prevent amyloidogenesis. Moreover, clearance of Abeta -apoE by endocytic apoE receptors such as LRP-1 and LRP-2 may also function to limit the extracellular level of complex as we have proposed for the Abeta -apoJ complex. In this regard, the endocytic action of LRP-1 and the recently described low density lipoprotein receptor family member termed apoER2 (24) may be more relevant to clearance of Abeta -apoE complex from brain parenchyma than LRP-2 since LRP-1 and apoER2 have widespread expression in the parenchyma (24-27), whereas LRP-2 expression in the brain is restricted to epithelial cells of the choroid plexus and ependyma (12, 28, 29).

Herein, evidence is presented indicating that one consequence of LRP-2-mediated endocytosis of Abeta -apoJ is that Abeta is targeted for lysosomal degradation. This is the end result for other LRP-2 ligands following their endocytosis (e.g. urokinase and plasminogen activator inhibitor-1 complex, low density lipoprotein, and apoJ (11, 30-32)). It may, however, seem paradoxical that Abeta could be degraded in lysosomes, considering that a number of studies indicate that it can be created in lysosomes as a result of proteolytic processing of amyloid beta -precursor protein (33, 34). It is possible that the lysosomal presentation of Abeta in the form of a complex with apoJ may make it more susceptible to proteinase degradation. It is important to note that LRP-2 is apparently not the only means by which extracellular Abeta can be internalized. For example, fibroblasts presumably lacking LRP-2 can internalize exogenously added 125I-Abeta 1-42 (35). This internalization pathway was shown to lead to intracellular accumulation of Abeta in the form of aggregates that are resistant to lysosomal proteinase degradation. LRP-2-mediated internalization may avoid this outcome by both preventing intracellular aggregate formation and promoting lysosomal degradation of Abeta .


FOOTNOTES

*   This work was supported by National Institutes of Health Grant DK45598 (to W. S. A.).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.
Dagger    Present address: Head and Neck Surgery Branch, Tumor Biology Section, NIDCD, NIH, 10 Center Dr., Bldg. 10, Rm. 5D55, MSC1419, Bethesda, MD 20892-1419.
§   To whom correspondence should be addressed: Medical University of South Carolina, Cell Biology and Anatomy Dept., 171 Ashley Ave., Charleston, SC 29425-2204. Tel.: 803-792-5482; Fax: 803-792-0664; E-mail: argraves{at}musc.edu.
1   The abbreviations used are: Abeta , amyloid beta -peptide; apoJ, apolipoprotein J/clusterin; LRP-2, low density lipoprotein receptor-related protein-2/megalin; RAP, receptor-associated protein; BSA, bovine serum albumin; TBS, Tris-buffered saline; mAb, monoclonal antibody; PBS, phosphate-buffered saline; dPBS, Dulbecco's PBS; PAGE, polyacrylamide gel electrophoresis; OG, N-octyl-beta -D-glucopyranoside; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; RA, retinoic acid; Bt2cAMP, dibutyryl cyclic AMP; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assays; SS, serum substitute.
2   LRP-2 is synonymous with glycoprotein 330 (gp330), brushin, and megalin.

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