The alpha 5beta 1 Integrin Mediates Elimination of Amyloid-beta Peptide and Protects Against Apoptosis

Michelle L. Matter,* Zhuohua Zhang,*Dagger Christer Nordstedt,§ and Erkki Ruoslahti*

* La Jolla Cancer Research Center, The Burnham Institute, La Jolla, California 92037; Dagger  Department of Neurobiology, Harvard Medical School, and Division of Neuroscience, The Children's Hospital, Boston, Massachusetts 02115; and § Department of Clinical Neuroscience, The Karolinska Hospital, Stockholm, Sweden S-171 76

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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The amyloid-beta peptide (Abeta ) can mediate cell attachment by binding to beta 1 integrins through an arg-his-asp sequence. We show here that the alpha 5beta 1 integrin, a fibronectin receptor, is an efficient binder of Abeta , and mediates cell attachment to nonfibrillar Abeta . Cells engineered to express alpha 5beta 1 internalized and degraded more added Abeta 1-40 than did alpha 5beta 1-negative control cells. Deposition of an insoluble Abeta 1-40 matrix around the alpha 5beta 1-expressing cells was reduced, and the cells showed less apoptosis than the control cells. Thus, the alpha 5beta 1 integrin may protect against Abeta deposition and toxicity, which is a course of Alzheimer's disease lesions.

    Introduction
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

INTEGRIN-mediated cell adhesion is necessary for the survival of many types of cells, and loss of adhesion causes apoptosis (reviewed in Frisch and Ruoslahti, 1997). The alpha 5beta 1 integrin may have a particularly prominent antiapoptotic effect because alpha 5beta 1 is the only integrin that protects cells from apoptosis in serum-free cultures (Zhang et al., 1995; O'Brien et al., 1996). alpha 5beta 1-mediated adhesion upregulates the antiapoptosis protein Bcl-2 (Zhang et al., 1995), and alpha 5beta 1 is one of a few integrins that activates the signaling protein Shc (Wary et al., 1996). These signaling events may partly explain its antiapoptotic effects.

beta 1 integrins have been shown to mediate cell adhesion to the amyloid beta (Abeta )1 protein, and alpha 5beta 1 has been proposed to be the integrin responsible for the Abeta binding (Ghiso et al., 1992). The amino acid sequence arg-his-asp (RHD) has been pinpointed as the integrin recognition site in Abeta (Ghiso et al., 1992; Sabo et al., 1995). This sequence resembles the general integrin recognition sequence RGD present in many extracellular matrix proteins (Ruoslahti, 1996a).

Abeta is a 39-42 amino acid protein derived from proteolytic cleavage of a larger membrane-spanning glycoprotein, the amyloid precursor protein (APP; Kang et al., 1987). Abeta forms fibrillar aggregates that can cause cell death by apoptosis (Loo et al., 1993; Pike et al., 1993; Lorenzo and Yanker, 1994). Enhanced deposition of Abeta matrix within the cortex, hippocampus, and vasculature of the brain correlates with neuronal cell death and ultimately dementia in Alzheimer's disease (AD; reviewed by Selkoe, 1994). Two predominant forms of Abeta (1-40 and 1-42) exist in AD that differ by two amino acid residues at the hydrophobic COOH terminus, a domain that is required for nucleation-dependent fibril formation (Jarret et al., 1993). The Abeta 1-40 form has a slower rate of fibril formation in vitro than the Abeta 1-42 form (Jarret et al., 1993).

There is evidence for three mechanisms of Abeta accumulation: overproduction of Abeta , production of longer forms of Abeta (which aggregate more), and impaired clearance of Abeta . The clearance pathways for fibrillar and soluble Abeta are incompletely known. Two cell surface receptors are known to bind Abeta . The scavenger receptor present on glial cells binds specifically to fibrillar Abeta , and appears to mediate clearance of small fibrillar Abeta aggregates in vitro (Paresce et al., 1996; Khoury et al., 1996). The receptor for advanced glycation end products binds both the soluble and fibrillar forms of Abeta , and may mediate some of the cytotoxic effects of fibrillar Abeta (Yan et al., 1996).

Because alpha 5beta 1 may also be an Abeta receptor, and because alpha 5beta 1 and Abeta have apparently contrasting effects on apoptosis, we sought to determine whether alpha 5beta 1 is indeed an Abeta -binding integrin and, if so, what effect it might have on the metabolism of Abeta and on cell survival. We show here that nonfibrillar Abeta binds to the alpha 5beta 1 integrin, and that this interaction promotes clearance of Abeta by cultured cells, reducing the formation of an insoluble Abeta fibrillar matrix and counteracting the toxic effects of the Abeta matrix. These results suggest a new function for alpha 5beta 1 as a binder of Abeta and a regulator of brain cell survival.

    Materials and Methods
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Materials & Methods
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Cells

The human neuroblastoma cell line (IMR-32) was obtained from the American Type Culture Collection (Rockville, MD). The CHO-B2 cells deficient in alpha 5beta 1 were from Dr. Rudolf Juliano (School of Medicine, University of North Carolina, Chapel Hill, NC; Schreiner et al., 1989). All cells were maintained in alpha -MEM (Sigma Chemical Co., St. Louis, MO) supplemented with 10% FBS and glutamine/pen-strep (Irvine Scientific, Santa Ana, CA). G418 (GIBCO BRL, Gaithersburg, MD) was added to the media of transfected cells at a concentration of 250 µg/ml.

Reagents

Amyloid beta 1-40 peptide (Abeta ) was synthesized as previously described (Nordstedt et al., 1994). Abeta was also purchased from a commercial source (Synthetic Amyloid Beta peptide 1-40; Bachem, Torrance, CA). Abeta 1-40 from both sources was examined for cell adhesion activity. Two out of the three Bachem lots tested showed adhesive activity (lots zn571 and wm365), while lot zn327 was not active. For water-free storage to prevent aggregation of Abeta into its fibrillar form, the peptide was dissolved and stored in 1,1,1,3,3,3,-hexafluoro-2-propanol (HFIP; Fluka Chemika, Neu-Ulm, Switerland). Before use, the peptide was lyophilized from HFIP, dissolved in sterile distilled water at 1 mg/ml, and tested immediately. The control peptide, Abeta 40-1, was purchased from Bachem, solubilized in water at 1 mg/ml, and tested immediately. Fibronectin was purchased from Chemicon International, Inc. (Temecula, CA), and vitronectin was purified as described (Yatohgo et al., 1988). Purified anti-human alpha 5 integrin monoclonal antibody (P1D6; Calbiochem-Novabiochem Corp., La Jolla, CA; Wayner et al., 1988) and purified mouse IgG (Sigma Chemical Co.) were used at a concentration of 50 µg/ml.

Transfection

The CHO-B2/alpha 5beta 1+, CHO-B2/alpha vbeta 1+, and IMR-32/alpha 5beta 1+ cells were generated by introducing cDNAs coding for the alpha 5 and alpha v integrin subunits into alpha 5beta 1-deficient CHO-B2 and IMR-32 cells (Schreiner et al., 1989; Bauer et al., 1992; Zhang et al., 1993, 1995). Transfectants expressing the integrin were cloned and expanded (Zhang et al., 1993; Zhang et al., 1995). CHO-B2 and IMR-32 control cells received the empty vector.

Integrin Analysis

Integrin expression of IMR-32 and CHO transfectants was analyzed by FACS using monoclonal antibodies against human alpha 5 (P1D6), alpha v (L230), and beta 1 (P4C10). FITC-conjugated goat anti-mouse antibody (Sigma Chemical Co.) was used as the secondary antibody. The same integrin antibodies were used to block integrin function in other experiments.

Cell Adhesion to Nonfibrillar Abeta 1-40

The cell attachment assay and the use of antibodies and peptides as inhibitors of adhesion have been described previously (Zhang et al., 1993; Matter and Laurie, 1994). Microtiter plates coated overnight at room temperature with nonfibrillar Abeta 1-40 peptide, control Abeta 40-1 peptide, or fibronectin were blocked with 1% BSA for 30 min at room temperature, the wells were rinsed once with PBS (pH 7.4), and cells were subsequently added (2 × 10 5 cells/well) in serum-free media and incubated for 60 min (37°C). Inhibition studies were performed by preincubating cells with antibody for 30 min (37°C; gentle agitation every 10 min), and then cells including antibodies were added to the coated wells. After a 60-min incubation at 37°C, plates were gently washed four times with PBS, fixed with 1% glutaraldehyde (Sigma Chemical Co.), PBS-washed once, stained with 0.5% crystal violet, 20% MEOH, washed under running distilled water, solubilized in 0.1 N sodium citrate, 50% ETOH, and read on an ELISA plate reader (Molecular Devices Corp., Sunnyvale, CA) using the 590-nm filter.

Adhesion assays with fibrillar Abeta 1-40 were performed as above. Before the adhesion assay, soluble Abeta 1-40 was incubated at 4°C for 96 h to allow self-aggregation of Abeta 1-40 into its fibrillar form (Jarret et al., 1993). Coating efficiency was measured by coating microtiter wells with either soluble [125I]Abeta 1-40 or preaggregated [125I]Abeta 1-40 at room temperature overnight. Nonbound peptide solution was removed, and the well and the nonbound peptide solution were counted. Both forms of Abeta 1-40 bound to the wells with an efficiency of ~70%.

Immunostaining of Abeta Fibrillar Matrix

Cells were plated on four-well PermanoxTM plastic slides (Nunc Inc., Naperville, IL) at 50,000 cells/well. 6 h after plating, the media was replaced with media containing Abeta 1-40 peptide (100 µg/ml) and incubated for 72 h at 37°C. The cultures were washed with PBS and fixed in PBS containing 3.7% paraformaldehyde and 10 mM sucrose, pH 7.4, for 30 min at room temperature. The cultures were then blocked with 1% BSA/PBS and stained with a polyclonal rabbit anti-human Abeta 1-40 peptide antibody (Chemicon International, Inc.) for 2 h, followed by goat anti-rabbit FITC-labeled IgG (Sigma Chemical Co.) secondary antibody. After antibody treatment, coverslips were mounted with Vectashield mounting medium (Vector Labs., Inc., Burlingame, CA) and analyzed under a fluorescent confocal microscope.

Analysis of Abeta in Matrix Deposition with Radiolabeled [125I]Abeta

125I-labeled Abeta 1-40 peptide was purchased as a lyophilized powder (25 µCi) from Nycomed Amersham, Inc. (Princeton, NJ). The powder was solubilized in sterile water and immediately added to 24-well culture dishes at a concentration of 2 ng/well. The specific activity of the 125I-labeled Abeta 1-40 peptide was 2 × 106 µCi/mmol.

Insolubilization of Abeta was analyzed using 125I-labeled Abeta 1-40 peptide as described previously for fibronectin matrix assembly (McKeown-Longo and Mosher, 1985; Morla and Ruoslahti, 1992). Cells were plated at 105 cells/ml (IMR variants) or 0.5 × 105 cells/ml (CHO variants) into 24-well tissue culture plates in media containing 10% serum. Media was replaced 6 h after plating with media containing [125I]Abeta 1-40 and 10% serum. Cells were cultured for 72 h at 37°C. The media was then removed, the wells were washed three times with PBS, and 5× SDS sample buffer (0.5M Tris pH 6.8, glycerol, 10% SDS, 0.5% bromophenol blue) was used to solubilize the [125I]Abeta matrix in each well.

For antibody inhibition experiments, cells were plated as above. 6 h after plating, the media was replaced with media containing the appropriate antibody and 10% serum. 125I-labeled Abeta 1-40 peptide (2 ng/well) was added to the antibody-containing media and incubated for 72 h at 37°C. The cells were then processed as above.

Internalization and Degradation of [125I]Soluble Abeta 1-40

Internalization of Abeta 1-40 added to cell layers was measured as described (Duckworth et al., 1972; McDermott and Gibson, 1997). Subconfluent cells were trypsinized and plated onto 24-well plates. Media was replaced 6 h after plating with [125I]Abeta 1-40 (2 ng/ml). The cells were incubated for 1 h with [125I]Abeta 1-40, the media was removed, cells were washed five times with PBS, and serum-containing media containing no Abeta 1-40 was added. The cells were cultured for 1 to 12 h at 37°C, washed three times with PBS, detached by EDTA, washed twice with PBS, lysed in 100 µl of 1% NP40 buffer for 10 min at 4°C, and lysate-analyzed for radioactivity.

For TCA precipitations, the cells were cultured for 72 h with [125I]Abeta 1-40 at 37°C, washed three times with PBS, detached by EDTA, washed twice with PBS, and lysed in 100 µl of 1% NP40 buffer for 10 min at 4°C. BSA/ PBS (100 µl, 1%) was added to the samples, the samples were vortexed, and 1.6 ml of TCA (12.5% wt/vol) was added with vortexing. The samples were centrifuged at 2,000 rpm for 10 min at 4°C, and the supernatant and pellet were collected for radioactive counting.

Secretion of 125I-Labeled Abeta 1-40

Subconfluent cells were detached with trypsin, washed once with media, and plated at 105 cells/ml in 24-well plates. 6 h after plating, media was replaced with 2 ng/ml of [125I]Abeta 1-40 in serum-containing media and incubated for 1 h at 37°C. The radiolabeled media was removed, and cells were washed five times in PBS before serum-containing media containing no Abeta was added to each well. At designated time points, 100 µl of media was collected, and [125I] was measured.

Apoptosis and Cell Viability Assays

The apoptotic effect of fibrillar Abeta was determined using the Apoptag Plus In Situ Apoptosis KitTM (Oncor, Inc., Gaithersburg, MD) that detects the 3'-OH region of cleaved DNA. Cells were plated on eight-chamber tissue culture glass slides (Miles Scientific Laboratories, Inc., Naperville, IL), and 6 h after plating the media was replaced with media containing either Abeta 1-40 peptide (50 µg/ml) or Abeta 40-1 control peptide (50 µg/ml) and 10% serum. Cells were cultured for 72 h at 37°C, and were then fixed in a solution containing 3.7% paraformaldehyde, 10 mM sucrose in PBS for 30 min at room temperature. Cells were stained following kit protocol, counterstained with propidium iodide/antifade solution (Oncor, Inc.), mounted, and viewed under a confocal microscope.

To measure apoptosis by nuclear fragmentation, cells were plated in wells coated with either 50 µg/ml of fibronectin, vitronectin, or Abeta 1-40 for 72 h in serum-free medium. Attached and floating cells were then collected by centrifugation, washed once with PBS, fixed with 3.7% paraformaldehyde for 10 min at room temperature, and stained with 0.1 µg of 4', 6-diamidino-2-phenylindole (DAPI) per ml in PBS. The stained cells were washed three times with PBS and mounted onto slides for analysis under a fluorescence microscope (Zhang et al., 1995).

Cell viability was assessed in several assays. The ability of cells to take up acridine orange/ethidium bromide was measured as described (Cotter and Martin, 1996). In brief, the assay was performed in 96-well tissue culture plates containing 100 µl media/well. Cells were plated in media containing 10% serum. 6 h after plating, the media was replaced with media containing various concentrations of the test reagents and 10% serum. The plates were incubated for 72 h at 37°C. At the 72-h time point, cells were trypsinized and resuspended in PBS at 0.5 × 106 cells/ml. 1 µl from a solution of acridine orange (100 µg/ml) and ethidium bromide (100 µg/ml) was added to a 25-µl cell suspension, incubated for 2 min at room temperature, and examined under 40× magnification using a Zeiss Fluorescence microscope.

Cells cultured in microtiter wells were pulsed with 25 µl of a 2.5 mg/ml MTT stock in PBS and incubated for 4 h. Then 100 µl of a solution containing 10% SDS, 0.01 N HCl was added, and the plates were incubated overnight (Tada et al., 1986). Absorption was read on a Vmax Microplate ReaderTM (Molecular Devices Corp., Sunnyvale, CA) using a reference wavelenth of 650 nm and a test wavelength of 590 nm. Test reagents were added to media alone in order to provide a blank.

To measure lactate dehydrogenase (LDH) release from cells, the colorimetric Cytotox 96-LDH-Release AssayTM (Promega Corp., Madison, WI) was performed according to the instructions of the manufacturer.

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The Integrin alpha 5beta 1 Mediates Cell Adhesion to Nonfibrillar Abeta 1-40

The RHD sequence in Abeta resembles the integrin recognition sequence RGD, and has been implicated in cell adhesion to Abeta via one or more of the beta 1 integrins (Ghiso et al., 1992; Sabo et al., 1995). We set out to determine which of the RGD-binding integrins bind to Abeta . A CHO cell line deficient in alpha 5 integrin subunit expression (CHO-B2) was transfected with cDNA encoding human alpha 5, alpha v, or vector alone, and was examined for its ability to adhere to a surface coated with Abeta 1-40. Each of the integrin transfectants adhered to Abeta in a dose-dependent manner, but cells that received the vector alone attached to Abeta within the BSA background range (Fig. 1 A). CHO-B2/alpha 5beta 1+ cells adhered strongly to Abeta , and CHO-B2/alpha vbeta 1+ cells were moderately adhesive, whereas the control cells CHO-B2/c did not adhere above BSA background levels. FACS analysis indicated that CHO-B2/alpha 5beta 1+ and CHO-B2/alpha vbeta 1+ cell transfectants were similar in their expression of the transfected integrin (Fig. 2, D and E). A control peptide in which the Abeta sequence is inverted (Abeta 40-1) did not have adhesive activity with any of the cell types tested (Fig. 1 B). In addition, integrin transfectants adhered only to soluble nonfibrillar Abeta 1-40, and not to fibrillar Abeta 1-40 (Fig. 1 B). Plates were coated with equal amounts of soluble and fibrillar Abeta 1-40 as measured by [125I]Abeta 1-40. The alpha 5beta 1-mediated cell adhesion to soluble Abeta 1-40 was inhibitable by the integrin-binding peptide GRGDSP, and by a function-blocking anti-alpha 5 integrin monoclonal antibody (P1D6; Fig. 1 C), but not by the control peptide GRGESP or a monoclonal antibody to alpha v (Fig. 1 C). The alpha vbeta 3 integrin, which also binds to RGD, does not mediate adhesion to Abeta because alpha vbeta 3-expressing IMR-90 cells did not adhere to Abeta when the alpha 5beta 1 and alpha vbeta 1 integrins were blocked with anti-alpha 5 and anti-beta 1 monoclonal antibodies (data not shown).


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Fig. 1.   Integrins mediate CHO cell adhesion to nonfibrillar Abeta . Adhesion of CHO cells to coated Abeta 1-40 was measured. The cells were transfected with human alpha 5 or alpha v integrin subunit cDNA. (A) The cells were seeded onto various concentrations of coated Abeta 1-40. (B) Cells were plated onto Abeta 1-40 coated in its soluble form, Abeta 1-40 coated after self-aggregation into its fibrillar form, or on the control peptide Abeta 40-1. (C) Cells were preincubated with either the blocking monoclonal antibodies to human alpha 5 (P1D6), human alpha v (L230), the integrin-binding peptideGRGDSP, or the control peptide GRGESP, and then seeded onto coated soluble Abeta 1-40. After a 60-min incubation at 37°C, attached cells were quantitated. Values represent the mean ± SD; n = 9.


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Fig. 2.   FACS analysis of alpha 5beta 1 integrin expression on IMR-32 neuroblastoma cells and CHO cells. alpha 5beta 1 surface expression by three IMR-32 cell clones transfected with human alpha 5 cDNA. IMR-32/ alpha 5beta 1 clone 4 (A), IMR-32/ alpha 5beta 1 clone 15 (B), and IMR-32/alpha 5beta 1 clone 16 (C; solid lines) is compared with a vector-transfected control line IMR32/c (A and B; dashed lines), and the parental cell line IMR-32/p (C; dashed line). (D) CHO cells transfected with the human alpha 5 cDNA express alpha 5beta 1 on their surface (solid line), whereas the vector-transfected control cells (CHO-B2/c) do not (dashed line). Cells were stained with a monoclonal antibody to the human alpha 5 integrin subunit, followed by an FITC-labeled secondary antibody, and analyzed by FACS. (E) CHO cells transfected with the human alpha v cDNA express alpha vbeta 1 on their surface (solid line), whereas the vector-transfected control cells (CHO-B2/c) do not. The staining was with a monoclonal antibody to the human alpha v integrin subunit.

We also tested the alpha 5-negative human neuroblastoma cell line IMR-32 (Neill et al., 1994) for Abeta attachment with (IMR-32/alpha 5beta 1+) and without (IMR-32/c) alpha 5 transfection (Fig. 2 A). Three separate clones were obtained that expressed human alpha 5beta 1 on their surface as detected by FACS analysis (Fig. 2, A-C). Each alpha 5beta 1-expressing clone adhered to coated Abeta 1-40 in a dose-dependent manner (Fig. 3 A), and cell adhesion was inhibitable by an anti-alpha 5 antibody (data not shown). The control-transfected IMR-32 cells (Fig. 2, A-C) attached poorly to this substrate (Fig. 3 A). Both the transfected and control cells attached well to vitronectin (data not shown), whereas the control peptide Abeta 40-1 and fibrillar Abeta 1-40 did not promote adhesion above BSA background levels for any of the IMR-32 cell lines (Fig. 3 B).


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Fig. 3.   Adhesion of IMR-32 cells to coated Abeta . (A) Adhesion of IMR-32 cells transfected with the human alpha 5 subunit IMR-32/ alpha 5beta 1 and vector-transfected IMR-32/c cells to Abeta 1-40 was measured as described in the legend for Fig. 1. (B) IMR-32 cell transfectants and control IMR-32/c and IMR-32/p cells were plated on Abeta 1-40 coated in its soluble or fibrillar form or on the control peptide Abeta 40-1, and cell adhesion was measured as described in the legend for Fig. 1. Values in A and B represent the mean ± SD; n = 9.

alpha 5beta 1 Reduces the Formation of an Insoluble Abeta Fibrillar Extracellular Matrix

An increase of insoluble Abeta fibrillar matrix is one hallmark of AD (Glenner and Wong, 1984; Masters et al., 1985). As shown above, the alpha 5beta 1 integrin bound to coated Abeta with the highest avidity among the integrins we tested. Therefore, we asked whether alpha 5beta 1 would affect the formation of an Abeta fibrillar matrix. Exogenous Abeta 1-40 added to cell cultures formed a matrix around the cells that was detectable by immunostaining with anti-Abeta antibodies. There was a substantial decrease in the formation of matrix from added Abeta in cultures of the alpha 5beta 1-expressing IMR-32 cell lines compared with the control lines (Fig. 4, A-D). Moreover, the matrix in the alpha 5beta 1+ cell cultures appeared to be cell-associated, whereas in the alpha 5beta 1- cell cultures it appeared to be largely independent of the cells.


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Fig. 4.   Immunofluorescent detection of reduced Abeta matrix in cultures expressing the alpha 5beta 1 integrin. Deposition of Abeta 1-40 matrix from soluble Abeta added to the culture media of IMR-32 control cells IMR-32/c (A) and IMR-32/p (B), and of alpha 5beta 1 expressing IMR-32 clones 4 (C) and 15 (D) was examined. The Abeta matrix was detected by a polyclonal anti-Abeta 1-40 antibody followed by a rhodamine-labeled secondary antibody. The experiment was repeated at least three times for each cell line, and representative results are shown. Bar, 50 µm.

To study quantitatively the formation of the Abeta matrix, the various IMR-32 lines were incubated with 125I-labeled Abeta for 72 h, and the amount of radiolabeled Abeta that had become soluble in detergent was measured. The IMR-32 clones expressing alpha 5beta 1 deposited fivefold less insoluble Abeta radioactivity than the control cells. Moreover, the P1D6 anti-alpha 5 antibody returned Abeta matrix formation in the alpha 5beta 1-expressing IMR-32 cultures to the level in the parental control cells (Fig. 5 A). A control antibody had no effect. CHO cells expressing alpha 5beta 1 also had less Abeta matrix than their control-transfected counterpart cells as judged from the insolubility of [125I]Abeta ; the difference was fourfold (Fig. 5 B). Adding the anti-alpha 5 antibody canceled the alpha 5beta 1 effect, but a control antibody did not. The insolubility of Abeta remained the same in the CHO control cell cultures regardless of the antibody added. These results indicate that cell expression of alpha 5beta 1 reduces Abeta matrix deposition threefold relative to the control cells. Because iodinated Abeta forms fibrils less readily than unlabeled Abeta 1-40 (Bush et al., 1994), it was not possible to use the [125I]Abeta to quantitate the proportion of the added Abeta 1-40 that becomes insolubilized.


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Fig. 5.   Quantitation of Abeta matrix deposition in cultures of IMR-32 cells and their alpha 5beta 1-expressing clones. (A) 125I-labeled soluble Abeta 1-40 was incubated with cultures of two control lines and three alpha 5beta 1-expressing lines at 37°C for 72 h in the presence of either the monoclonal antibody to the human alpha 5 integrin subunit (P1D6) or control IgG. The total amount of 125I-labeled Abeta 1-40 associated with an SDS soluble matrix was determined as described in Materials and Methods. The reduction of Abeta 1-40 matrix deposition in the alpha 5beta 1-expressing cultures was reversed by an anti-alpha 5 integrin monoclonal antibody (P1D6), but not by the control IgG. (B) CHO-B2/alpha 5beta 1 cells show reduced deposition of [125I] to Abeta matrix relative to control CHO cells. The experimental procedure was the same as in A. The P1D6 antibody reversed the alpha 5beta 1-dependent matrix reduction, whereas the control mouse IgG had no effect. Experiments in A and B were repeated at least four times, and representative results are shown. Values represent the mean ± SD; n = 3.

Soluble Abeta 1-40 is Taken Up By Cells and Partially Degraded Via an alpha 5beta 1-Mediated Pathway

Possible reasons for the alpha 5beta 1-mediated reduction of Abeta matrix include internalization of soluble Abeta 1-40, degradation of the peptide, or both. Neuronal cells have been shown to internalize Abeta , but the mechanism for this internalization is only incompletely known (Ida et al., 1996, Hammad et al., 1997). To investigate the possibility that binding alpha 5beta 1 to soluble Abeta initiates cellular uptake of Abeta , we examined the processing of 125I-labeled Abeta 1-40 by alpha 5beta 1+ and alpha 5beta 1- cells. Initially, CHO-B2/c control cells and transfectants were incubated for 1 h with [125I]Abeta 1-40, and were then examined for cell-associated radioactivity. The alpha 5beta 1-expressing CHO-B2 cells contained twofold more radioactivity at 1 and 12 h than the control CHO-B2/c cells (Fig. 6 A).


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Fig. 6.   alpha 5beta 1-expressing cells take up and degrade soluble Abeta . (A) Cell-associated 125I-labeled Abeta 1-40 in CHO-B2/c control cells and alpha 5beta 1-expressing CHO-B2 cells. Cell cultures were incubated for 1 h with soluble 125I-labeled Abeta 1-40, detached with EDTA, and solubilized with NP40 representing both cell-associated and internalized Abeta . (B) Degradation of 125I-labeled Abeta 1-40 in IMR-32 control cells and alpha 5beta 1-expressing clones. The cultures were incubated with soluble 125I-labeled Abeta 1-40 over a 72-h period, and cells were detached with EDTA, solubilized with NP40, and the NP40 extracts were precipitated with trichloroacetic acid (TCA) to separate 125I-labeled Abeta into a degraded soluble fraction and an insoluble fraction representing intact or partially degraded Abeta . (C) Cell-associated 125I-labeled Abeta 1-40 in CHO control cells (CHO-B2/c) and alpha 5beta 1-expressing clones (CHO-B2/alpha 5beta 1). (D and E) Release of [125I] from 125I-labeled Abeta 1-40 into cell culture medium. 125I-labeled soluble Abeta 1-40 was added to the culture media of various IMR-32 (D) or CHO (E) cell lines and incubated for 1 h. After that, the media was removed, the cell layer was washed, and new media was added. The release of radioactivity into the media was monitored over a 72-h period. Experiments in A-E were repeated at least three times, and representative results are shown. Values in A-E represent the mean ± SD; n = 3.

Cell cultures were then incubated with 125I-labeled Abeta over a 72-h period to determine whether the [125I]Abeta taken up by the cells was degraded. alpha 5beta 1-expressing IMR-32 cells contained twofold more radioactivity after the 72-h incubation than alpha 5beta 1-negative IMR-32 cells (Fig. 6 B). Part of the radioactivity was soluble in TCA, indicating that Abeta had been degraded. CHO cells internalized and degraded soluble Abeta in a similar manner, with alpha 5beta 1-expressing cells containing eightfold more TCA-soluble radioactivity than alpha 5beta 1-negative cells (Fig. 6 C). The CHO cells expressing alpha 5beta 1 bound 10% of the added Abeta , whereas the control cells bound only 0.4%. Moreover, 90% of the cell-associated Abeta was degraded in the CHO-alpha 5beta 1 expressers. The higher expression levels of alpha 5beta 1 on the CHO transfectants (Fig. 2, D and E) may explain why these cells bound and internalized more radiolabeled Abeta than the IMR-32 transfectants.

We next examined whether Abeta was released into the culture medium. The release of radioactivity into cell culture media was monitored over a 72-h period that followed a 1-h incubation with 125I-labeled Abeta 1-40. The media of alpha 5beta 1-expressing IMR-32 and CHO cells contained twofold more radioactivity than the corresponding control cell media (Fig. 6, D and E). These results point to an alpha 5beta 1-dependent pathway that internalizes and degrades Abeta .

alpha 5beta 1 Protects Cells Against Abeta Induced Apoptosis

Having established an alpha 5beta 1-dependent mechanism for the inhibition of Abeta matrix deposition, we examined whether the reduction of the Abeta matrix would promote neuronal cell survival in cultures treated with Abeta . IMR-32 cell lines cultured with exogenous soluble Abeta 1-40 underwent apoptosis in the absence of alpha 5beta 1 (Fig. 7, A and B), but three alpha 5beta 1-expressing lines did not (two are shown in Fig. 7, C and D). The control peptide Abeta 40-1 caused no apoptosis in the control (Fig. 7, E and F) or alpha 5beta 1-expressing cells (not shown). Analysis of acridine orange/ethidium bromide uptake revealed three times more apoptosis in the control cells than in the IMR-32 alpha 5beta 1-expressers (Fig. 8).


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Fig. 7.   Effect of Abeta matrix on apoptosis. Expression of alpha 5beta 1 protects IMR-32 neuroblastoma cells from apoptosis induced by Abeta matrix formation. Cell cultures were incubated for 72 h with Abeta 1-40 to allow aggregation of Abeta into a matrix, and the cultures were then examined for evidence of cytotoxicity by using the TUNEL assay. A percentage of IMR-32/c (A) and IMR-32/p (B) cells underwent apoptosis (green). The viable cells (red) are counterstained with propidium iodide. No apoptotic cells were seen in IMR-32/alpha 5beta 1 transfectant clones 4 (C) and 15 (D) under the same conditions. (E-F) The IMR-32/c and IMR-32/p control cells remained viable when incubated for 72 h with the inactive control peptide Abeta 40-1. These experiments were repeated at least three times for each cell line, and representative results are shown. Bar, 50 µm.


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Fig. 8.   Effect of Abeta matrix on apoptosis. Expression of alpha 5beta 1 protects IMR-32 neuroblastoma cells from apoptosis induced by Abeta matrix formation. Cell cultures were incubated for 72 h with Abeta 1-40 to allow aggregation of Abeta into a matrix, and the cultures were then examined for evidence of apoptosis by measuring the ability of cells to take up acridine orange/ethidium bromide. A higher percentage of alpha 5beta 1-IMR-32/c and IMR-32/p cells than alpha 5beta 1+ transfectants underwent apoptosis. Values represent the mean ± SD; n = 9.

We also assessed the Abeta effect by using the MTT assay, which measures cell viability by detecting the ability of a mitochondrial enzyme to reduce its substrate. Abeta -treated IMR-32 control cells lost their ability to reduce MTT in a manner that was dependent on the dose of Abeta , whereas Abeta had almost no effect on the alpha 5beta 1-expressing cell lines (Fig. 9 A). The control peptide Abeta 40-1 had no effect on MTT reduction in any of the cell types, even at the highest test concentration (Fig. 9 B). To examine further the cytotoxicity of Abeta 1-40, we used an assay that measures the release of LDH upon cell lysis (Behl et al., 1994). A threefold increase in LDH levels relative to controls was seen in the alpha 5beta 1- IMR-32 cells cultured in the presence of Abeta 1-40, whereas Abeta 1-40 had no effect on the LDH levels of the alpha 5beta 1+ cells (Fig. 10 A). These results indicate that alpha 5beta 1-mediated Abeta binding protects the IMR-32 cells from the cytotoxicity of aggregated Abeta , presumably by inhibiting its aggregation into fibrils. No apoptosis was caused by Abeta in any of the CHO cell lines, as examined by TUNEL staining, the MTT assay, and the LDH assay, indicating that these cells are resistant to the cytotoxic effects of an Abeta matrix.


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Fig. 9.   Effect of Abeta 1-40 on cell viability. Cell survival in cultures containing aggregated Abeta . Cell cultures were incubated for 72 h with Abeta 1-40 to allow aggregation of Abeta into a matrix, and the cultures were then examined for their ability to reduce MTT. (A) The control cells---IMR-32/c (circles) and IMR-32/p (triangles)---lost the ability to reduce MTT, whereas the IMR-32/alpha 5beta 1 clones 4 (diamonds) and 15 (squares) were essentially resistant under the same conditions. Values represent the mean ± SD; n = 9. (B) The control peptide Abeta 40-1 did not affect the ability of any cell type to reduce MTT.


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Fig. 10.   Cell survival in cultures containing aggregated Abeta . Cell cultures were incubated for 72 h with Abeta 1-40 to allow aggregation of Abeta into a matrix, and the LDH levels within the cultures were analyzed. (A) The alpha 5beta 1- cells IMR-32/c and IMR-32/p showed increased LDH levels, whereas the IMR-32/alpha 5beta 1 clones 4, 15, and 16 maintained LDH levels similar to those seen in the presence of the Abeta 40-1 control peptide. Values represent the mean ± SD; n = 9. (B) Cell survival on Abeta coated from freshly made solution onto a plastic surface. Cells were seeded onto Abeta 1-40, fibronectin, and vitronectin coated onto microtiter wells, cultured in serum-free conditions, and examined by DAPI staining after 96 h. The alpha 5beta 1 transfectants remained viable on Abeta 1-40 and fibronectin, but not on vitronectin.

We previously demonstrated that cell attachment through alpha 5beta 1 protects CHO cells from apoptosis when cultured in a serum-free environment (Zhang et al., 1995). Therefore, we examined whether ligation of alpha 5beta 1 to coated Abeta 1-40 would protect alpha 5beta 1-expressing CHO cells from apoptosis in serum-free cultures. CHO-B2/alpha 5beta 1+ cells were plated on either fibronectin, vitronectin, or Abeta -coated dishes and examined for survival 96 h after serum withdrawal. CHO-B2/alpha 5beta 1+ cells survived on Abeta and fibronectin, whereas cells plated on vitronectin underwent apoptosis (Fig. 10 B). These results indicate that alpha 5beta 1 can also protect cells from apoptosis by mediating cell attachment to coated Abeta .

    Discussion
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We report that the alpha 5beta 1 integrin mediates cell adhesion to Abeta and promotes internalization and degradation of Abeta . This alpha 5beta 1-Abeta interaction correlates with both an increase in the clearance of soluble Abeta , a reduction in the formation of an insoluble Abeta fibrillar matrix, and a decrease of the toxicity of Abeta to cells. This study provides one mechanism for regulating Abeta accumulation.

Our data, showing that Abeta binds to alpha 5beta 1, and to a lesser extent alpha vbeta 1, is in agreement with previous reports that Abeta mediates cell attachment, and that the RHD sequence in it serves as an integrin-binding site (Ghiso et al., 1992; Sabo et al., 1995). The RHD sequence apparently functions as a mimic of the RGD sequence in fibronectin, the matrix ligand of alpha 5beta 1 (Ruoslahti, 1996a), because a short peptide containing the RGD sequence inhibits Abeta binding. alpha 5beta 1 binds only to nonfibrillar Abeta , since we did not see any detectable cell adhesion to aggregated fibrillar Abeta . Therefore, other receptors presumably mediate cellular interactions with fibrillar Abeta , and are responsible for the cytotoxic effects of this form of Abeta . The alpha 5beta 1 integrin is one of the most discriminating of the RGD-directed integrins with regard to its ligand specificity (Ruoslahti, 1996b). In addition to its main ligand fibronectin, the alpha 5beta 1 integrin has only been shown to bind to the bacterial protein invasin (Watari et al., 1996) and the insulin-like growth factor binding protein IGFBP-X (Jones et al., 1993). Our results add Abeta among its ligands. The binding site for alpha 5beta 1 seems to be available only in Abeta , not in its precursor protein (APP; B. Bossy, M.L. Matter, and E. Ruoslahti, unpublished results).

The alpha 5beta 1 integrin may play a role in the rapid clearance of Abeta that occurs in the normal brain (Ghersi-Egea et al., 1996). We show that expression of the alpha 5beta 1 integrin is associated with increased cellular uptake and degradation and decreased matrix deposition of Abeta in cell cultures. Moreover, reversal of this effect with a function-blocking anti-alpha 5 antibody established a causal link between alpha 5beta 1 activity and increased clearance of Abeta . Although more complex explanations of this effect are possible, the binding of Abeta to alpha 5beta 1 shown here suggests that Abeta binds to alpha 5beta 1 at the cell surface, and is subsequently internalized into a cellular compartment where it is degraded. This hypothesis is in agreement with previous results showing that a neuronal cell line internalizes Abeta from culture medium in a manner that is dependent on the NH2 terminus of Abeta where the RHD sequence resides (Ida et al., 1996). The lipoprotein Apo J can also reduce the formation of fibrillar Abeta by causing it to be internalized and degraded (Hammad et al., 1997). Thus, it is likely that more than one mechanism plays a role in the regulation of Abeta accumulation in vivo. Clearly, a transgenic animal expressing the amyloid precursor protein with a mutated RHD sequence would be of great interest in testing the contribution of the RHD sequence and integrin-binding to the metabolism of Abeta .

The alpha 5beta 1 integrin circulates through the endocytic cycle (Bretscher, 1989; Bretscher, 1992). Inhibiting exocytosis with primaquin causes accumulation of internalized alpha 5beta 1 in an intracellular pool that returns to the cell surface over time. Recent studies have shown that internalization of fibrillar Abeta promotes accumulation of stable fibrillar Abeta in the late endosome/secondary lysosome compartment, whereas internalization of soluble Abeta leads to degradation of the peptide in the same compartment (Knauer et al., 1992; Koo and Squazzo, 1994; Yang et al., 1995). This result is in agreement with our data, showing that soluble Abeta is internalized through an alpha 5beta 1 integrin-mediated pathway, and is at least partially degraded, presumably within endosomes. Thus, clearance of soluble Abeta can be mediated by the alpha 5beta 1 integrin, presumably through the receptor-mediated endocytosis pathway that normally internalizes this integrin.

alpha 5beta 1 may play a protective role in the brain by suppressing Abeta cytotoxicity. We provide evidence for two separate mechanisms that could be responsible for such a protective effect. First, we show that alpha 5beta 1-mediated adhesion to nonfibrillar Abeta protects cells from apoptosis in cell culture. Upregulation of Bcl-2 (Zhang et al., 1995) and activation of the MAPK pathway (Wary et al., 1997) may be responsible for this pathway. The second and potentially more important mechanism is suggested by our demonstration that alpha 5beta 1 suppresses the apoptotic effects of Abeta by reducing production of toxic Abeta matrix.

The alpha 5beta 1 integrin and alpha vbeta 1 are present in the adult central nervous system (Grooms et al., 1993). Immunostaining for alpha 5beta 1 shows that it is expressed in the vasculature, cortex, and hippocampus of adult rat brain (Bahr et al., 1991; Pagani et al., 1992; Tawil et al., 1994; for review see Sargent Jones, 1996). Moreover, primary hippocampal neurons express alpha 5beta 1 (Yamazaki et al., 1997). Soluble Abeta 1-40 is present in vivo (Seubert et al., 1992), and is rapidly cleared when injected into normal rats (Ghersi-Egea et al., 1996). Our results suggest that alpha 5beta 1 may mediate the clearance of Abeta , and that alpha 5beta 1 may play a significant role in protecting the brain from the Abeta -initiated pathology that in its extreme form causes AD.

    Footnotes

Received for publication 2 December 1997 and in revised form 25 February 1998.

   The present address of Z. Zhang is Department of Neurobiology, Harvard Medical School, The Children's Hospital, Enders 260, 300 Longwood Ave., Boston, MA 02115.
   Address all correspondence to Erkki Ruoslahti, The Burnham Institute, 10901 North Torrey Pines Rd., La Jolla, CA 90237. Tel.: 619-646-3125; Fax: 619-646-3199; E-mail: ruoslahti{at}burnham-inst.org

We thank Drs. Blaise Bossy, Eva Engvall, and Kristiina Vuori for comments on the manuscript, and Dr. Edward Monosov for help with the confocal microscopy. This work was supported by grant CA28896 (E. Ruoslahti) and Cancer Center Support Grant CA 30199 from the National Cancer Institute, Department of Health and Human Services. M.L. Matter is supported by postdoctoral training grant CA09579 from the National Institutes of Health.

    Abbreviations used in this paper

Abeta , amyloid beta  peptide; AD, Alzheimer's disease, LDH, lactate dehydrogenase; RHD, arg-his-asp.

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