extracellular matrix regulates the amount of the β-amyloid precursor protein and its amyloidogenic...

JOURNAL OF CELLULAR PHYSIOLOGY 166360-369 (1996)

Extracellular Matrix Regulates the Amount of the B-Amyloid Precursor Protein and Its

Amyloidogenic Fragments FRANCISCA C. BRONFMAN, CLAUD10 SOTO, LUClA TAPIA, VERONICA TAPIA,

AND NIBALDO C. INESTROSA’ Departamento de Biologfa Celular y Molecular, Facultad de Ciencias Biologicas,

Pontificia Universidad Cat6lica de Chile, Santiago, Chile

We have studied the influence of the extracellular matrix (ECM) on the amount of P-amyloid precursor protein (APP) and C-terminal amyloid-bearing fragments in 3T3 fibroblasts. After incubation with ECM components, the cellular APP content of 3T3 cells changed. Besides, different substrata including collagen, fibronectin, laminin, vitronectin, and heparin, determined changes in the amount of a C-terminal 22 kDa-fragment. The regulation of amyloidogenic fragments by the ECM was transient; in fact, when 3T3 cells were plated on tissue culture dishes coated with collagen or vitronectin, maximal levels of the 22 kDa fragment were observed 12 h after plating; in the presence of fibronectin, the maximum level of the amyloidogenic fragment was obtained 36 h after plating. These results indicate that the ECM modulates in a transient way the generation of APP-derived polypeptides containing the amyloid-P-peptide (AP). The ECM does not have a generalized effect on 3T3 fibroblasts, because no significant differences in cell attachment, growth rate, whole-cell polypeptide pattern, PI integrin and a-tubulin levels were observed on cells grown on various matrix proteins. Laminin, collagen, and heparin also influence the level of an amyloidogenic fragment of 35 kDa in Neuro 2A neuronal cells, without a significant change in the neuronal marker acetylcholinesterase. In this case, however, a long-lasting response to ECM molecules was observed. These observations provide evidence that ECM molecules influence APP biogenesis, including the generation of amyloidogenic fragments containing the AB peptide. Our studies might prove significant to understand the localized increment of P-amyloid deposition in selected areas of the brain of Alzheimer’s patients. o 1996 Wiley-Liss, Inc.

The expression and processing of the p-amyloid precursor protein (APP) is of intense current interest (Selkoe, 1994), since the discovery that mutations in APP cause Alzheimer’s disease (AD) (Hardy, 1993; Mullan and Crawford, 1993). APP constitutes a family of different isoforms that are produced by alternative splicing (Selkoe, 1994). The amino acid sequences of these APP isoforms show characteristic features of typ- ical transmembrane glycoproteins (Kang et al., 1987; Soto et al., 1994). Major secreted forms of APP are generated by proteolytic cleavage from the cell surface into the extracellular space (Esch et al., 19901, and eventually they are incorporated into the extracellular matrix (ECM) (Klier et al., 1990; Small et al., 1992). In addition to the secretory pathway, APP is also pro- cessed and degraded within an endosomalflysosomal pathway (Estus et al., 1992; Busciglio et al., 1993; Si- man et al., 1993). Full-length APP can be internalized from the cell surface and targeted to endosomes and lysosomes, where COOH-terminal fragments containing the entire amyloid-P-peptide (AP) sequence have been detected (Golde et al., 1992; Haass et al., 1992a). Recently a precursor-product relationship be- 0 1996 WILEY-LISS, INC.

tween APP carboxy-terminal fragments and the AP peptide has been demonstrated (Higaki et al., 1995). The biological functions of AP peptide could be regulated by complexing with ECM molecules (Koo et al., 1993).

Many efforts have been made to identify the functional significance of APP in various biological pro- cesses, including cell adhesion (Breen et al., 1991), cell proliferation (Ninomiya et al., 1993; Alvarez et al., 19951, calcium regulation (Mattson et al., 1993), tro- phism (Whitson et al., 19891, toxicity (Yankner et al., 1989; Yoshikawa et al., 19921, demyelination (Alvarez et al., 1995), axonal sprouting and neurite outgrowth (Alvarez et al., 1992; Allinquant et al., 1995). It is known that APP can bind to different molecules of the cell environment such as components of the ECM (Brandan and Inestrosa, 19931, including laminin (Kib- bey et al., 1993) and heparan sulfate proteoglycans

Received October 31, 1994; accepted August 8, 1995. *To whom reprint requestidcorrespondence should be addressed at Molecular Neurobiology Unit, Pontificial Catholic University of Chile, P.O. Box 114-D, Santiago, Chile.

ECM INFLUENCES APP 361

(Narindrasorasak et al., 1991; Snow et al., 1994). These results suggest that APP may be part of an adhesion/ transduction system involved in cell-cell and or cell- matrix interactions (Allinquant et al., 1995).

Cell responsiveness to growth and transforming factors is often controlled by components of the ECM (Lin and Bissell, 1993; Lander and Calof, 1993; Clark and Brugge, 1995). APP biogenesis itself is affected by a variety of substances like transforming growth factor P, interleukin-lp, and heparin-binding growth factors, including basic fibroblast growth factor (Schubert et al., 1989; Gray and Patel, 1993). Although adhesive interactions of APP with components of the ECM have been studied in detail (Breen et al., 1991; Kibbey et al., 1993; Small et al., 1994), little is known about the effect of ECM molecules on APP and COOH-terminal amyloid-bearing fragments levels.

It is well known that the interaction between cells and ECM plays a crucial role in regulating tissue-specific functions (Hynes and Yamada, 1982; Inestrosa, 1988; Hynes and Lander, 1992). In fact, the ECM can profoundly influence cell growth, metabolism, gene expression, and differentiation of several cell types (Rei- chardt and Tomaselli, 1991; Lander and Calof, 1993; Lin and Bissell, 1993). Abnormalities in any of the ECM components or their integrin receptors could cause some of the alterations observed in AD (Fillit and Lev- eugle, 1995), like the reduced adhesiveness of skin fibroblasts (Ueda et al., 1989).

The present study was undertaken to investigate whether ECM molecules could regulate the amount of APP and its processing to amyloidogenic fragments. We report here that ECM components induce a change in APP content and in COOH-fragments containing the AP peptide levels in both fibroblasts and neuronal cells.

MATERIALS AND METHODS Cell culture conditions

1. Mouse Balb/c 3T3 (American Type Culture Collec- tion, Rockville, MD; CCC 163) fibroblasts were grown in Dulbecco's modified Eagle's medium (DMEM) (Sigma, St. Louis, MO), containing fetal bovine serum (Sigma), and 1% (v/v) penicillin-streptomycin solution (Sigma). Fibroblasts were maintained at 37°C in a hu- midified atmosphere of 95% air and 5% C02 . All experiments were carried out between 80-90% confluence and the same pool of cells was used for the control and experimental points. The passage number was between 12 and 15. In some experiments, the cell growth rate was measured indirectly by counting the number of cells recovered from each tissue culture dish after expo- sure to different matrices.

2. Mouse C1300 neuroblastoma cells, Neuro 2A (Breen et al., 1991), were maintained in DMEM medium containing 10% (v/v) fetal calf serum, 100 U/ml penicillin, 2.5 mg/ml amphotericin B, 100 mg/ml streptomycin,and 3.7 g/L NaHC03 (pH 7.4).

Antisera The polyclonal antibodies anti-GID (residues 175-

186 of AF'P molecule) and anti-COOH (amino acids 649-675) were a gift of Dr. Gregory Cole, University of California, San Diego (Cole et al., 1989). The polyclonal antibody anti-C8 (residues 676-695 of AF'P) (Selkoe et al., 1988) and antibody 1282 (residues 1-40 of AP

peptide) (Haass et al., 1992a) were a gift of Dr. Dennis J . Selkoe of the Harvard Medical School, Boston. The monoclonal antibody 4G8 directed against residues 17- 24 of AP (Kim et al., 1990) was a gift of Dr. Nikolaos Robakis of the Mount Sinai Medical Center, New York, and of Drs. Kwang Kim and Henryk Wisniewski of the New York State Institute for Basic Research in Devel- opmental Disabilities, Staten Island, New York. The polyclonal antibody SGY 2134 against the Apl-40, was a giR of Dr. Stephen G. Younkin from Mayo Clinic, Jacksonville, FL. Monoclonal antibody against anti-cc- tubulin was purchased from Sigma. Finally the monoclonal antibody AIIB2 directed against P l integrin was a kind gift of Dr. Caroline Damsky from the University of California, San Francisco.

Preparation of matrices

Tissue culture substrates were prepared under ster- ile conditions by coating Petri dishes (Nunc) 8 h at room temperature with the following molecules obtained from Sigma. Collagen type I11 and n7; fibronectin, laminin, vitronectin, and poly-D-lysine were used at 1 pgl cm', concentration in which these molecules are biologi- cally active (Charo et al., 1987; Cheresh and Spiro, 1987; Tomaselli et al., 1987; Needham et al., 1988; Breen et al., 1991). In the case of heparin, a glycosami- noglycan not present in the matrix, a higher concentration was used (600 pg/cm'), because the active matrix component, a heparan sulfate proteoglycan, was not available to us.

Analysis of APP and amyloid-bearing fragments

Cells were removed from the culture flasks by incubation with 0.15 M phosphate-buffered saline, pH 7.4, containing 10 mM EDTA and counted in the presence of Trypan Blue, using a Neubauer chamber. To obtain cellular proteins, cells (4 x lo5) were harvested and homogenized in 10 mM Tris-HC1, 0.1% Triton X-100 plus protease inhibitors (Inestrosa et al., 1988), and then centrifuged at 15,000 rpm for 15 min in an Ep- pendorf centrifuge. Total protein was determined by the method of Lowry et al. (1951), and used to adjust equal amounts of supernatant loaded in SDS-gels. In each experimental case, 100 kg of protein was resolved by SDS-polyacrylamide gel electrophoresis (SDS- PAGE) using the Laemmli system (Laemmli, 1970).

To detect the amyloidogenic fragments of APP, low molecular weight peptides were separated by Tris-Tric- ine 16% SDS-PAGE (Schagger and von Jagow, 1987; Estus et al., 1992). After electrophoresis, the proteins were transferred electrophoretically to nitrocellulose membranes (Towbin et al., 1979). Non-specific binding of primary antibody was blocked by incubating the blots with 5% non-fat dry milk in TS buffer (10 mM Tris- HCl, pH 8.0, 150 mM NaC1, 0.01% NaN3).

After incubation with specific antibodies against APP and AP peptide (1:500) overnight a t 4"C, immunoreactive bands were detected with an alkaline phosphatase- conjugated anti-rabbit or anti-mouse IgG (Sigma) using 4-chloro-1-naphthol and 5-bromo-4-chloro 3-indolyl phosphate (both from Sigma). Quantitation of individ- ual bands was performed by densitometric scanning at 550 nm.

362 BRONFMAN ET AL

A

APP-

B

7t

C d H1 PD FH C

Fig. 1. APP amount in cells grown on different ECM molecules. 3T3 fibroblasts (85-90% confluence) were grown on tissue culture dishes coated with different ECM molecules for 48 h at 37°C. Then the medium was removed and the dishes were washed with PBS. The attached cells were removed by incubation with PBS-EDTA and counted as indicated in Materials and Methods. Polypeptides present in cell extracts were separated in 10% SDS-PAGE and immunoblotted with anti-GID antibody directed against the N-terminal region ofAF'P (Cole et al., 1989). A: Immunoblots of cells grown on different matrices. The substrates used to grow the cells are indicated in the lower part of the figure. B Densitometric evaluation of the blot shown in (A). The results presented in this figure were repeated more than 4 times, with a similar pattern. The densitometric analysis is the mean I SEM of 3 experiments. The P values for the densitometric analysis are for plastic (C) compared with collagen (Coll) P < 0.05, vitronectin (VN) P < 0.005, fibronectin (FN) P < 0.001, and poly-D-lysine (PD) P < 0,001 (Student's t-test). AU the substrates were used a t a concentration of 1 pgkm".

Analysis of p1 integrin and a-tubulin The amount of a-tubulin and p l integrin present in

the cells incubated on different matrices was determined, after 10% SDS-PAGE (Laemmli, 1970). One hundred micrograms of protein of each group of cells were transferred t o nitrocellulose membranes (Towbin et al., 19791, as described above. A dilution of was used with the monoclonal anti-a-tubulin and a $j dilution with the monoclonal antibody AIIB2 directed

0 5 10 3 5 20 25 30

TIME (min) Fig, 2. Cell adhesion of 3T3 cells grown on different ECM components. 3T3 cells were removed from the culture flasks by incubation with 0.15 M phosphate buffered saline (pH 7.4) containing 10 mM EDTA. Then the cells were added to 6 well plates coated with ECM molecules (see Fig. 1) at a density of 5 x lo5 cells per well and incubated for different periods of time at 37°C. The bound and unbound cells were counted in the presence of Trypan Blue. The adhesivity of the cells was calculated as the percentage of the cells attached on each matdcel l attached to a control (100%). Plastic, 0; poly-D-lysine, A; vitronectin, 0; fibronectin, 0; collagen type 111, A.

against p l integrin. Immunoreactive bands were detected with an anti-mouse IgG.

Acetylcholinesterase assay Acetylcholinesterase, an enzyme present in NeuroM

neuroblastoma cells (Stieger et al., 1989), was measured by the method of Ellman et al. (1961), as described previously (Inestrosa et al., 1981, 1982).

Whole-cell polypeptide pattern Forty micrograms of protein of each sample was sepa-

rated in 10% SDS-PAGE (Laemmli, 19701, and then stained with Coomassie blue.

RESULTS APP content in 3T3 cells grown on different

ECM components To study the influence of ECM molecules on the

amount of cellular APP, we cultivated equal amounts of 3T3 fibroblasts on tissue culture dishes coated with different substrates. To determine the amount of APP level, we performed Western blot analysis of cell lysates with a polyclonal anti-NH2 antiserum (Cole et al., 1989). On a qualitative basis, differences in full-length APP 751-770 content were clearly observed among fibroblasts incubated for 2 days on different substrates. Particularly interesting was the case of cells plated on fibronectin (see also poly-D-lysine), where increasing

ECM INFLUENCES APP 363

Fig. 3. Morphology of 3T3 cells grown on different ECM molecules. Fibroblasts were grown as described in Materials and Methods and incubated on different ECM molecules for 48 h at 37°C. as described in Figure 2. Cells were at 90% confluence. A Fibroblasts grown on

uncoated plastic dish. B Cells grown on collagen type 111. C 3T3 cells grown on fibronectin. D Cells grown on poly-D-lysine. E: Fibroblasts grown on vitronectin.

amounts of APP 751-770 were observed in comparison with cells plated on collagen and on uncoated dishes. Likewise, intermediate values were obtained for cells grown on vitronectin (Fig. U). The qualitative conch- sions for the different matrices are strengthened by the

densitometric evaluation of several Western blots (Fig. 1B). These results suggest that the amount of full- length APP 751-770 is modulated by ECM components.

The above results were obtained under conditions in which the ability of cells to adhere to different subtrates

364

A a b C

C COII VN

d e

BRONFMAN ET AL.

FN PD

Fig. 4. Whole-cell polypeptide pattern, p 1 integrin and a-tubulin leveh in 3T3 cells cultivated on different substrates. A: Polypeptides of 3T3 fibroblasts cultivated on different substrates (indicated in the lower part of the figure) were separated in a 10% SDS-PAGE. In each case 40 pg of protein was loaded per lane. The gel was stained with Coomassie Blue. Control (C); collagen (Coll); vitronectin 6"); fibro- nedin (FN) and poly-D-lysine (PD). B Cells incubated on different matrices for 48 h were collected, homogenized and aliquots of 100 pg/ protein were separated in a 10% SDS-PAGE. Then, the polypeptides

B

was similar (Fig. 2). The cell's growth rate was also similar in all matrices, and the general cell health and development of 3T3 fibroblasts was normal. Cells showed a slightly different morphology when cultivated on different ECM-coated plastic dishes (Fig. 3); in some matrices fibroblasts assume a spindle-shaped morphology (collagen and poly-D-lysine) in contrast with cells grown on plastic dishes (Fig. 3A, vs. B and D). As a control for the specificity of the effect of ECM over the APP content, the whole-cell polypeptidic pattern was studied in 3T3 cells incubated on different substrates. As Figure 4A shows, the same polypeptidic pattern was observed in cells grown on different matrices. To fur- ther explore the specificity of the matrix effect on APP, the levels of a membrane-bound protein, j.31 integrin (Fig. 4B), and a microtubular component, a-tubulin (Fig. 4C), were also evaluated. Results indicate that the levels of both control proteins remained unaltered in 3T3 cells incubated on different ECM substrates. These results indicated that the ECM does not have a generalized effect on cell growth, cytoskeletal or surface proteins, but a selective effect on APP. Amyloidogenic derivatives of APP in 3T3 cells

grown on different ECM components To evaluate the presence of the amyloidogenic deriva-

tives of APP, we used a monoclonal antibody (4G8)

-0, lntegrin

a b c d . e

C -Tu bul in

a b c d e were immunoblotted using as primary antibody a monoclonal antibody directed against p 1 integrin (If10 dilution, overnight), a kind gifi of Dr. Caroline Damsky, UCSF. C Same as (B), but the nitrocellulose paper was incubated with a monoclonal antibody against a-tubulin (1/500, overnight) (Sigma). In (B) and (C) the letters in each lane correspond to the substrates indicated in (A). Results are representative of a t least five experiments for the whole-cell polypeptidic pattern, and at least two experiments with anti p 1 integrin and anti a-tubulin.

raised against a synthetic peptide corresponding to residues 17-24 of AP (Kim et al., 1990). On Western blots antibody 4G8 recognized fragments of 35, 22, and 12 kDa on 3T3 fibroblasts (Fig. 5A). The same pattern was observed with antibodies against the C-terminal region of APP and the AP peptide, but not with antibodies specific for the N-terminal of APP (data not shown). Among the amyloid-bearing fragments, the 22 kDa polypeptide appears to be the more abundant. When the amyloidogenic derivatives of APP were examined in fibroblasts grown on different substrates, including laminin and heparin, an interesting pattern was observed. Even at short times of incubation (30 min) differences were noticed in the reactivity of the AP-bearing fragment of 22 kDa (Fig. 5B). Clearly the cells plated on collagen, fibronectin, and laminin contain higher amounts of this polypeptide than cells incubated on vitronectin, heparin, or plastic. On the contrary, 3T3 fibroblasts incubated on poly-D-lysine exhibit the lowest reactivity for this fragment.

The effect of ECM components on the amyloidogenic derivatives of APP was also evaluated after 2 days of incubation. At this time no difference was observed in the relative amounts of the amyloidogenic fragments of APP (data not shown). However, when the time course of the appearance of the amyloidogenic APP derivatives was studied in cells plated on collagen, a vari-

A 1

ECM INFLUENCES APP

2

66 KDa-

-35 KDa

-22 KDa

-12 KDa

4 KDa-

a b Ab 4G8

B -35 KDa

-22 KDa

-1 2 KDa

C Coll LAM FN Fig. 5. Level of amyloidogenic fragments of APP in 3T3 cells cultivated on different substrates. Reactivity pattern of antibody 4G8 in fibroblast lysates. A: Proteins were separated in 16% Tris-Tricine SDS-PAGE as described in Materials and Methods. (1) The separation of selected standard proteins. Lane a, SDS-VII-L kit; and lane b, SDS- 17s kit, both from Sigma. (2) Immunoblot analysis of lysates of 3T3 cells using as primary antibody the anti-4G8 directed against amino acids 17-24 of the Ap peptide (Kim et al., 1990). The star indicates the 22 kDa fragment, the most abundant amyloidogenic peptide in 3T3 cells. B: Fibroblasts were incubated, for 30 min at 37"C, in tissue culture dishes coated with different ECM molecules at a concentration

365

HEP VN PD of 1 pg/cm2, except for heparin, where 0.6 mg/cmz was used (see Mate- rials and Methods). Cells were collected, homogenized, and aliquots (100 pg protein) were separated in 16% Tris-Tricine SDS-PAGE. Then the polypeptides were immunoblotted using a monoclonal antibody directed against residues 17-24 of the Ap peptide (antibody 4G8). At the right part of the figure the molecular mass of the three main amyloidogenic fragments is indicated. Results are representative of the three experiments carried out. The error was less than 15%. C, control; CoU, collagen; LAM, laminin; FN, fibronectin; HEP, heparin; VN, vitronectin; PD, poly-D-lysine.

ation in the amount of the 22 kDa fragment was observed. In fact, the amyloidogenic fragment becomes accumulated between 8 and 24 h, with a maximum level at 12 h and decays thereafter (Fig. 6A). The 35 kDa-band did not change during the same period of time. The time course of appearance of the 22 kDa amyloidogenic fragment was different depending of

ric evaluation of the Western blots obtained from cells plated on different ECM molecules. For vitronectin (and also collagen) the maximal amount was obtained at 12 h and for fibronectin at 36 h after plating. Intermediate values were observed for cells plated on poly-D-lysine (data not shown). These results show-that the transient accumulation of the 22 kDa amyloidogenic derivative of APP observed in 3T3 the-substiate used: Figure 6B shows the dksitomet-

366 BRONFMAN ET AL.

A

-35 KDa

-22 KDa

4 8 12 24 36 48 T I M E ( h )

12 24 36 48 0 8 TIME (hours)

Fig. 6. Time course of the appearance of amyloidogenic peptides in 3T3 cells. A: Fibroblasts grown on collagen-coated dishes were incubated for the times indicated below each figure. At each time point, the level of the 35 and 22 kDa fragments was studied by immunoblot- ting with an antibody against the COOH-terminal domain of APP (Cole et al., 1989). B Densitometric evaluation of the time course of appearance of the 22 kDa amyloidogenic fragment in 3T3 cells cultivated on fibronectin (Fn) and vitronectin (Vn). In each case a representative experiment of the three carried out is shown.

cells depends on the specific ECM component used as cell substrate.

Fragment which contains the Afl peptide is regulated by ECM components in neuronal cells

In order to study whether the ECM regulates the amyloidogenic fragments of APP, in other cell types, the Neuro 2A neuronal cells were selected. In such neurons the cell adhesion t o both laminin and collagen IV (but not collagen type 111) is only slightly higher than for cells grown on uncoated plastic tissue culture dishes

(Breen et al., 1991). Therefore, we plated Neuro 2A cells on laminin and collagen lV. Heparin was used as an additional substrate to compare with our previous studies on fibroblasts. After 48 h of incubation, the A& containing fragments were evaluated using an antibody directed against the residues 1-40 of the AP peptide (Estus et al., 1992). As Figure 7 indicates, a 35 kDa fragment increased in those cells plated over ECM molecules. Cells plated on collagen and laminin contain a higher amount of the amyloidogenic fragment than neuronal cells incubated on heparin. Only a small amount was observed in cells plated on uncoated plastic tissue culture dishes (Fig. 7B). As a control for the specificity of the effect of ECM molecules over the 35 kDa fragment of APP, the whole-cell polypeptidic pattern was studied in neuronal cells incubated on different substrates. Figure 7A shows no indication of a van- ation in the whole-cell polypeptidic pattern. A neuronal marker, the enzyme acetylcholinesterase (Inestrosa et al., 1981, 1987) was also studied in the Neuro 2A cells (Stieger et al., 1989). As Table 1 indicates, no change was observed in cells cultivated on laminin or collagen compared with cells grown on plastic. A small decrease in enzymatic activity was detected in cells incubated on heparin.

DISCUSSION The ECM has profound effects on cells: it can influ-

ence cell shape, proliferation, motility, differentiation, growth factor responsiveness, and gene expression. The ECM glycoproteins that elicit these responses, include laminin, fibronectin, vitronectin, and collagens (Rei- chardt and Tomaselli, 1991; Hynes and Lander, 1992). In the present study, the ability of ECM molecules to influence the expression level of the full-length APP and its processing to AP-containing derivatives was demonstrated in both fibroblasts and neuronal cells.

The purpose of this investigation was to determine whether the interactions of fibroblasts or neuronal cells with their surrounding ECM are important for the regulation of APP and APP fragments. Within this context, the results obtained with laminin, fibronectin, and collagen on the time course of appearance of a 22 kDa fragment bearing the AP peptide in 3T3 cells were particularly interesting. This amyloidogenic fragment of APP has been proposed as an intermediary derivative in the APP processing pathway which generates AP in transfected human 293 cells (Knops et al., 1992), and has been also described as a stable fragment containing the AP peptide in meningeal microvessels and other human brain regions (Tamoaka et al., 1992). Previous studies have shown a marked enrichment of a 22 kDa fragment of AF'P in leupeptin-treated cells, suggesting that this fragment may be an intermediary in the endo- somal-lysosomal pathway of AP production (Haass et al., 1992b; Selkoe, 1994). Our results suggest that the ECM modulates the level of APP and its amyloidogenic peptides. These effects could be due to a direct regulation of APP expression by ECM signaling molecules and/or to a modulation of APP processing by the ECM- related cytoskeletal network in the cytoplasmic com- partment (Clark and Brugge, 1995).

A potentially important observation is the fact that 3T3 cells grown on poly-D-lysine had the largest amounts of APP and the lowest amounts of an amy-

ECM INFLUENCES APP

A B KDa

367

- 35 KDa 20,

14 - C HEP LAM Coll C HEP LAMCol l

Fig. 7. Level of an amyloidogenic fragment of APP in neuronal cells incubated on different substrates. A: Polypeptides obtained from Neuro 2Aneuronal cells cultivated for 48 h on laminin (LAM), collagen IV (Coll) (both 1 pg/cm2) and heparin (HEP) (0.6 mg/cm2) were separated in 16% Tris-Tricine SDS-PAGE (40 proteidane) and stained with Coomassie Blue. B In parallel experiments, the samples were separated by SDS electrophoresis and the polypeptides immunoblot-

ted with an antibody against the A&.4o peptide (antibody SGY), a kind gift of Dr. Steve Younkin. Pre-incubation of the antibody with synthetic AB1.40 peptide (Chiron Corp., Emeryville, CA) eliminated almost all the immunostaining of the 35 kDa fragment in Neuro 2A cells, incubated either with laminin (LAM), collagen (Coll), or heparin (HEP). Results are representative of at least four experiments carried out. C, control.

TABLE 1. Acetylcholinesterase activity of N e w 2A cells grown on different extracellular matrix (ECM) molecules'

Acetylcholiesterase activity Type of substratum (mU/mg protein)

Plastic 6.60 2 0.22 Collagen IV 7.40 f 0.24 Laminin 5.74 f 0.31 Heparin 4.95 * 0.36* 'Neuronal cells were grown on different ECM molecules and on uncoated tissue culture dishes for 48 h at 37°C. *The acetylcholinesterase activity of cells grown on heparin is significantly different from cells grown on plastic, P < 0.001, Student's t-test. Results were the mean I SEM of three experiments performed in triplicate. mu corresponds to milliUnits of ecetylcholinesterase activity (nrnol acetylthiocholine iodide hy- dnAyzed/min/mg protein).

loidogenic fragment of APP. The opposite is true for fibroblasts grown on collagen type 111. This latter result is interesting because collagen type I11 synthesis has been shown to decline during in vitro and in vivo aging of human skin fibroblasts (Takeda et al., 1992). Per- haps, some de-regulation or alterations within ECM molecules might favor the generation of the amyloidogenic fragments of APP. In this context, the fact that a precursor-product relationship has been demonstrated between the amyloidogenic fragments of APP and the AP peptide (Higaki et al., 19951, opens the possibility that some disorder of the ECM may be involved in the pathogenesis of AD. On different grounds, a similar idea has been recently formulated (Fillit and Leveugle, 1995).

The regulation of the amyloidogenic fragments of APP by the ECM also occurs in neuronal cells. This is an interesting finding because it has been hypothesized that neurons are the richest source of AP in brain (Ya- mazaki et al., 1995). As in the case of 3T3 cells, laminin and heparin were also able to influence the amount of

an amyloidogenic fragment of APP in neuronal cells. These results suggest that the effect of the ECM molecules in the accumulation of cellular APP and the si- multaneous regulation of APP-specific COOH-terminal fragments is a widespread phenomenon, and might be relevant to the pathogenesis of AD. In neuronal cells, the effect of ECM molecules on the amyloidogenic fragments of APP was observed after a few days of incubation; therefore in contrast to what happens with 3T3 fibroblasts, a long-lasting response to ECM molecules was present in neuronal cells.

Under the broadest definition of ECM-insoluble material found between cells-must be included those extracellular deposits that occur in certain disease states (Lander and Calof, 1993). The amyloids are a large group of such deposits, and they are characterized by a relatively uniform histological appearance, even though their molecular composition may be quite different depending on the disease process involved (Lans- bury, 1992). Amyloidosis involving the nervous system is a key feature of AD, and also of the so-called prion encephalopathies, like CreutzfeldJacob disease (Jar- rett and Lansbury, 1993). As it turns out, the similarit- ies between amyloids and normal ECM go beyond the mere fact that both are insoluble. For example, evidence indicates that proteoglycans are common, possi- bly invariant, components of amyloid deposits (Snow et al., 1994; Fillit and Leveugle, 1995).

The APP and particularly the amyloidogenic polypeptides are related to the pathogenesis of AD (Hardy, 1993; Selkoe, 1994). Moreover the @ aggregation is involved in the formation of amyloid fibrils, which con- stitute the core of the senile plaques (Soto et al., 1994, 1995). Such plaques occur in specific regions of the brain related to memory and cognition, including the basal forebrain, hippocampus, limbic and association

BRONFMAN ET AL 368

cortices (Hyman et al., 1986; Katzman and Saitoh, 1991). Although at the present time, there is no satis- factory explanation for the selective plaque deposition and neuronal death that occurs in AD (Kosik, 1992; Ma et al., 1994), it is known that the formation of senile plaques begins with diffuse AP deposits in the extracellular spaces of the brain, which then mature by increasing the aggregation of the AP plus several molecules, including ECM components (Brandan and Inestrosa, 1993; Fillit and Leveugle, 1995). The fact that ECM influences the biogenesis of APP and the level of amyloidogenic derivatives containing the AP peptide, may be relevant to amyloid deposition. In this context, it has been reported that the distribution of some ECM molecules is altered in the brain of AD patients. For example laminin, a neurite outgrowth and neuronal migration promoting agent (Calof et al., 1991), is increased in the hippocampus of AD patients (Kowall and McKee, 1990). It is also of interest to point out that neuronal degeneration of the brain induced the expression of ECM proteins like laminin (Liesi et al., 1984). Additional studies concerning the influence of the ECM on the expression and processing of neuronal APP are important in order to understand the biological basis of amyloid deposition in the brain of AD patients. Dur- ing the revision of this paper, the influence of ECM molecules on the biogenesis of APP and its C-terminal fragments has been independently confirmed in BV-2 cells a microglial cell line (Monning et al., 1995).

ACKNOWLEDGMENTS We thank Drs. Enrique Brandan and Jorge Garrido

for their comments and help with the manuscript. We also thank Drs. Gregory Cole, Kwang Kim, Nikolaos Robakis, Dennis Selkoe, Steve Younkin, and Caroline Damsky for their kind gift of antibodies. The contribu- tion of Juan Godoy, Verbnica Palma, and Pablo Figue- roa during the early phases of this work is gratefully acknowledged. This research was supported by grant 1940694 from FONDECYT t o N.C.I., a postdoctoral fellowship from CONICYT and Fundaci6n Andes to C.S., and a research fellowship from DIUC and a predoctoral fellowship from CONICYT to F.C.B.

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