©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Extracellular Matrix Influences the Biogenesis of Amyloid Precursor Protein in Microglial Cells (*)

(Received for publication, February 7, 1994; and in revised form, October 27, 1994)

Ursula Mönning(§) (1) Rupert Sandbrink (§) Andreas Weidemann (§) Richard B. Banati (2) Colin L. Masters (3) Konrad Beyreuther (§)

From the  (1)Center for Molecular Biology Heidelberg, University of Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Federal Republic of Germany, the (2)Department of Neuromorphology, Max Planck Institute for Psychiatry, D-82452 Planegg-Martinsried, Federal Republic of Germany, and the (3)Department of Pathology, University of Melbourne, Parkville, Victoria 3052, Australia

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

During axotomy studies, we discovered that the betaA4-amyloid precursor protein (APP) participates in immune responses of the central nervous system. Since microglia constitute the main immune effector cell population of this response, we used the murine microglial cell line BV-2 to analyze immune response-related APP expression. We show that interaction of microglia with the extracellular environment, particularly components of the extracellular matrix, affects APP secretion as well as intracellular APP biogenesis and catabolism. Fibronectin enhanced APP secretion and decreased the level of cellular mature transmembrane APP, whereas laminin and collagen caused a decrease in secretion and an accumulation of cellular mature APP and APP fragments.

Our results demonstrate that APP plays a fundamental role in the regulation of microglial mobility, i.e. migration, initial target recognition, and binding. The decrease in APP secretion and the concomitant increase in cellular mature APP were accompanied by an accumulation of C-terminal APP fragments. Enrichment of APP and APP fragments is assumedly based on inhibition of catabolic processes that is caused by a disorganization of the actin microfilament network. These observations provide evidence that microglia, which are closely associated with certain amyloid deposits in the brain of Alzheimer patients, can play a key role in initial events of amyloidogenesis by initiating accumulation of APP and also of amyloidogenic APP fragments in response to physiological changes upon brain injury.


INTRODUCTION

Alzheimer's disease is the most common cause of dementia in the elderly. The most prominent neuropathological features of this disease are intracerebral and cerebrovascular amyloid deposits (for reviews, see (1) and (2) ). The major proteinaceous component of these deposits is the betaA4-peptide that is generated by proteolytic cleavage from a parent amyloid protein precursor (APP). (^1)

APP constitutes a family of different isoforms that are produced by alternative splicing. The major and ubiquitous primary translation products consist of 695, 751, and 770 amino acid residues (APP695, APP751, and APP770, respectively)(3, 4, 5, 6) . In addition, we identified additional APP transcripts lacking exon 15, which leads to an exclusion of 18 amino acids, generating isoforms with 677, 733, and 752 amino acid residues, respectively. These ubiquitously expressed isoforms were termed L-APP (L-APP677, L-APP733, and L-APP752) according to their first identification in human peripheral mononuclear leukocytes including microglia/brain macrophages(7, 8, 9, 10) .

The amino acid sequences of these APP/L-APP isoforms show characteristic features of typical transmembrane glycoproteins(3) . Secreted forms of APP are generated by proteolytic cleavage within the amyloidogenic region(11, 12) . Recent reports have shown that soluble betaA4-peptides can be released in the extracellular milieu upon proteolytic breakdown of transmembrane APP. The betaA4-peptide was identified in media of neuronal and non-neuronal cell cultures as well as in body fluids of Alzheimer patients and of controls. Hence, it was suggested that the cleavage of APP into betaA4 is a normal, nonpathological event and does not cause amyloid deposition(13, 14, 15) .

Many efforts have been made to identify the functional significance of APP in various biological processes. It is known that APP can bind to different molecules of the cell environment such as components of the extracellular matrix (ECM)(16, 17, 18, 19) . It has also been shown that APP contains a sequence that has growth-promoting activity(20) . The identification of a domain with homology to the serine protease inhibitor of Kunitz type II points to a serine protease inhibitor function of APP. The soluble form of APP, secreted by platelets, is identical to protease nexin II, the natural inhibitor of blood coagulation factor XIa(21, 22, 23, 24, 25) . Since multiple biologically active sites have been identified, it has been proposed that APP plays a role in the regulation of diverse biological processes including inflammation, immune response, regeneration, wound healing, neuronal development, and axonal growth(8, 26, 27, 28, 29) .

APP/betaA4 biogenesis itself is affected by a variety of substances like cytokines, mitogens, and neurotransmitters(30, 31, 32) . Although adhesive interactions of APP with components of the extracellular matrix were studied in detail, little is known about the effect of extracellular matrix molecules on APP biogenesis.

ECM is a complex network that is composed of an array of macromolecules. Interactions of a cell with its surrounding ECM are important for the regulation of cell function and tissue architecture (33) . It is known that cells of the immune system show rapid and extensive physiological changes upon adherence to ECM. Monocytes/macrophages have proven to be an especially valuable system to study protein expression in response to ECM molecules (for a review, see (34) ). Brain macrophages, called microglial cells, were shown to resemble tissue macrophages(35) . They are the main immune effector cell population of the brain(35, 36, 37) . The number of activated microglial cells in brain increases under various neuropathological conditions, such as trauma and inflammation(35, 38, 39) . Neuronal re/degeneration are also associated with the expression of extracellular matrix proteins like fibronectin and laminin(40, 41) . This may suggest that ECM plays a crucial role in morphological transformation of microglia and, consequently, in differentiation of microglial cells(42) .

Since a close association between microglia and amyloid deposits could be demonstrated(43) , it has been postulated that microglial cells play an important pathological role in amyloidogenesis of Alzheimer's disease(44) . The immortalized microglial cell line BV-2 shares several of the features characteristic of activated microglia in vivo, such as antigen profile, phagocytic capacity, and antimicrobial activity(45) . The characteristics of this cultured mouse microglial cell line with respect to APP biosynthesis provide useful information on the possible function of APP during immune reactions of the central nervous system. We have studied the influence of components of ECM on BV-2 microglial APP biogenesis. We show that the interactions of microglia with ECM affect APP secretion as well as the intracellular biogenesis of APP, thus regulating APP metabolism and amyloidogenicity.


MATERIALS AND METHODS

Cell Line and Cell Culture

The microglial cell line BV-2 (45) was cultured permanently in Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum (Life Technologies, Inc.) and 2 mM glutamine. For preparation of substratum-coated dishes, plates with a diameter of 60 mm were incubated for 12 h at 4 °C with 20 µg/ml fibronectin, 20 µg/ml laminin, 100 µg/ml polylysine, or 100 µg/ml collagen type I, respectively. To set up experimental cultures on ECM-coated dishes, BV-2 microglial cells were removed by scraping and seeded in suspension at 5 times 10^5 cells/ml in 3.0 ml of medium consisting of optiMEM (Life Technologies, Inc.) and 2% fetal calf serum. Colchicine (2 µM), cytochalasin B (5 µM), cytochalasin D (2 µM), and nocodazole (5 µM) (all from from Sigma) were added to the experimental cultures for 12 h.

Immunoprecipitation and Immunoblotting

Conditioned medium was removed from the cell cultures and cleared by centrifugation. Cells were collected by scraping in 1.5 ml of phosphate-buffered saline and were washed once with phosphate-buffered saline. For lysis, the cells (1.5 times 10^6) were resuspended in 0.4 ml of lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 1% Triton X-100, 2 mM phenylmethanesulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1.6 mg/ml iodoacetamide) and incubated for 30 min on ice. Cell lysates were centrifuged at 10,000 times g for 5 min. The resulting supernatants were diluted with an equal volume of ice-cooled phosphate-buffered saline and subjected to immunoprecipitation with the polyclonal anti-CT antiserum raised against the cytoplasmic part of APP(46) . Conditioned medium was supplemented with 0.1 volume of medium buffer (1 M Tris (pH 8.0), 100 mM EDTA, 10% Nonidet P-40, and 100 mM phenylmethanesulfonyl fluoride) and precipitated with the polyclonal anti-FdAPP antiserum(46) . Precipitation and analysis were done as described previously(47) .

For analysis of C-terminal APP fragments, 50 µl of chloroform/methanol-precipitated cell lysates were fractionated on a 12.5% Tris/Tricine gel (48) and subjected to immunoblotting. Detection of C-terminal APP fragments was done with affinity-purified anti-CT IgG.

Cell Transfection

For transient expression of human APLP2/763 and APP695, monolayers of COS-7 cells were transfected with 20 µg of cytomegalovirus expression vector encoding APLP2 and APP695 cDNA, respectively, using Lipofectin reagent (Life Technologies, Inc.) according to the manufacturer's instructions. Analysis of APP and APLP2 expression was done 48 h after transfection by immunoprecipitation and chloroform/methanol precipitation followed by 7% SDS-gel electrophoresis and immunoblotting using the monoclonal antibody 22C11.

Isolation of RNA, Northern Hybridization, and Reverse Transcription-Polymerase Chain Reaction

Total RNA preparation and Northern hybridization with APP cDNA and glyceraldehyde-3-phosphate dehydrogenase cDNA restriction fragments were done as described previously(47) . 2 µg of RNA were reverse-transcribed with 200 units of Super-Script (Life Technologies, Inc.) using oligo(dT) as primer following the manufacturer's protocol. Amplification of cDNA preparations was performed by polymerase chain reaction(50) . Oligonucleotide primer and reaction conditions have been described in detail(10) .


RESULTS

Analysis of APP Biosynthesis in the Microglial Cell Line BV-2

For molecular characterization of APP expression in BV-2 cells, we performed a Western blot analysis of cell lysate and conditioned medium after immunoprecipitation with a polyclonal anti-CT antiserum (lysate) or a polyclonal anti-FdAPP antiserum. As shown in Fig. 1a, the transformed microglial cells expressed APP isoforms containing the C terminus in the predominant molecular mass ranges of 95-130 and 140-145 kDa, corresponding to all described major translation products of the APP gene (APP695, APP751, and APP770 and L-APP677, L-APP733, and L-APP752). However, they represent predominantly immature and mature Kunitz protease inhibitor-containing transmembrane APP isoforms, as can be deduced from a comparison with the lymphoid cell line H9(8) . In conditioned medium of microglial cells, secretory APP isoforms in a broad molecular mass range were identified (Fig. 1a; see also Fig. 3a). The recently described APP-like protein, APLP2(65, 66) , was not precipitated under the performed precipitation conditions with the polyclonal anti-CT and anti-FdAPP antisera (Fig. 1b, IP, lane3). However, direct analysis of chloroform/methanol-extracted cell lysate and conditioned medium by immunoblotting with the monoclonal antibody 22C11 revealed APLP2-specific bands (Fig. 1b, direct, lane3). For comparison, the analysis was concomitantly done with cell lysate and conditioned medium of nontransfected (lane2) and APP695-transfected (lane1) COS cells. Full-length APLP2 comigrated with endogenous APP; secreted APLP2 comigrated with secreted APP695.


Figure 1: Molecular characterization of APP expression in the microglial cell line BV-2. Cells were cultivated for 12 h on plastic culture plates in optiMEM supplemented with 2% fetal calf serum. a, APP biosynthesis of microglial cells. Cell lysates and conditioned media were subjected to immunoprecipitation with anti-FdAPP. The precipitates were analyzed by 7% SDS-PAGE followed by Western blot analysis using the monoclonal antibody 22C11 (dilution of 1:10,000). b, specificity of anti-APP antisera. Monolayers of COS cells were transfected with cDNAs of APP695 (lane1) and APLP2/763 (lane3). Half of the cell lysates and conditioned media of nontransfected COS cells (lane2) and COS transfectants were then subjected to immunoprecipitation (IP) with the anti-CT or anti-FdAPP antiserum; the other half was precipitated by chloroform/methanol extraction (direct). Analysis of precipitated proteins was performed by 7% SDS-PAGE followed by immunoblotting using the monoclonal antibody 22C11. c, alternative splicing of primary APP transcripts in BV-2 cells. Amplified cDNA fragments correspond to APP770, APP751/L-APP752, L-APP733, APP695, and L-APP677 mRNAs.




Figure 3: Effect of different ECM proteins on APP biosynthesis. a, Western blot analysis of APP isoforms precipitated from cell lysates and conditioned media of BV-2 cells. Microglial cells were cultivated for 16 h on different substrates (culture plate plastic, polylysine, fibronectin, laminin, and collagen type I). Subsequently, APPs from cells and media were immunoprecipitated with the anti-FdAPP antiserum, subjected to 7% SDS-PAGE, and analyzed by Western blotting using the monoclonal antibody 22C11. b, determination of total amounts of APP precipitated from cell lysates and conditioned media of BV-2 cells. Determination was done by densitometric scanning of the Western blot presented in a. The total amounts of APP were calculated from the amounts of cellular APP and of APP detected in conditioned medium. The amounts are shown in densitometric units. c, comparison of relative amounts of cellular immature and mature APPs and secretory APP. The relative amounts of APP detected in cell lysates (immature and mature transmembrane) and APP detected in conditioned media (secretory) of microglial cells cultivated on plastic, polylysine, fibronectin, laminin, and collagen are shown graphically. Determination was done as described for b (in each case, the total amounts of densitometric units are equivalent to 100%).



The alternative splicing of primary APP transcripts in BV-2 cells was investigated by quantitative reverse transcription-polymerase chain reaction analysis (10) as shown in Fig. 1c. Microglial cells expressed more than two-thirds of their APP mRNA as exon 7 (Kunitz protease inhibitor)-containing transcripts: 22% of total APP mRNA represented APP770 mRNA, 45% APP751/L-APP752 mRNA (polymerase chain reaction products unresolved), and 25% L-APP733 mRNA. Polymerase chain reaction products corresponding to APP695 and L-APP677 mRNAs were detected in relatively low amounts of total mRNA (each 4%). The results described above were obtained for microglial cells cultivated on plastic culture dishes.

Morphology and Adhesive Properties of Microglial Cells Cultured on Different Matrices

To investigate adhesive and phenotypic properties of BV-2 cells under different extracellular conditions, we cultivated equal amounts of microglial cells on tissue culture plates coated with different substrates (plastic, polylysine, fibronectin, laminin, and collagen type I). Their phenotypic behavior is presented in Fig. 2. Microglial cells grown on a plastic substratum were characterized by both spindle- and round-shaped morphologies. BV-2 cells spread on fibronectin and polylysine showed a marked increase in extending processes associated with an increased adhesiveness. Attachment was strongest to fibronectin. The observed microglial morphology seemed to be an intermediate stage between the two classical microglial phenotypes observed in vivo, i.e. ``amoeboid'' and ``ramified'' microglia(64) . BV-2 cells quickly lost their characteristic morphology after being plated on plastic dishes precoated with laminin. Here, an increased amount of cells showed weak adhesiveness with a round and small cell morphology lacking filopodial projections. Instead, cell aggregates could be observed. Similarly, on collagen type I-coated tissue plates, microglial cells grew in suspension by forming big cell aggregates without any adhesiveness to the plates.


Figure 2: Phenotypic behavior of the microglial cell line BV-2 grown on different substrates. Phase-contrast micrographs were photographed after 20 h of cultivation in fetal calf serum-reduced medium (magnification times 50). a, cell culture plastic; b, polylysine (100 µg/ml); c, fibronectin (20 µg/ml); d, laminin (20 µg/ml); e, collagen type I (100 µg/ml).



Influence of ECM on APP Biosynthesis

Because considerable phenotypic changes occur in microglial cells cultured on different substrates, we examined the capacity of cells cultured on these matrices to synthesize and secrete amyloid precursor proteins. Analysis of total microglial APP gene expression under different cultivation conditions was done by Northern blotting. The overall rate of protein synthesis of different cultivated cells was estimated by analysis of glyceraldehyde-3-phosphate dehydrogenase gene expression. RNA bands were quantitated by use of a PhosphorImager. The ratio of APP to glyceraldehyde-3-phosphate dehydrogenase reflects specific APP expression. As shown in Table 1, microglia cultivated on plastic plates exhibited the lowest biosynthetic activity, but the highest ratio of APP to glyceraldehyde-3-phosphate dehydrogenase expression. Although the biosynthetic activity of BV-2 cells was increased by cultivation on ECM-coated plates, a 50-70% decrease in the ratio of APP to glyceraldehyde-3-phosphate dehydrogenase was observed.



For analysis of APP metabolism, microglial cells coated on plastic, polylysine-, fibronectin-, laminin-, or collagen-treated dishes were subjected to immunoprecipitation followed by immunoblotting. The results are shown in Fig. 3a. In cell lysates, immunoreactive bands in the molecular mass ranges of 95-130 and 140-145 kDa were observed, but to different extents. In conditioned medium, secretory isoforms in the molecular mass ranges of 90-100 and 115-125 kDa were revealed.

The division of total APP in cellular immature and mature APPs as well as secretory APP under different cultivation conditions is summarized graphically in Fig. 3c. The relative amounts (in percent) were calculated from the total amount of APP shown in Fig. 3b. Microglia cultivated on plastic culture dishes produced mainly secreted APP. Secretory APP represented nearly 80% of total APP. Only low amounts of cellular immature APP isoforms were visible. The level of APP secretion from cells cultivated on the artificial matrix polylysine was about 10% lower than of microglial cells cultivated on plastic. Instead, a 10% increase in cellular APP was observed. Increased amounts of cellular APP and a concomitant decrease in APP secretion were pronounced in microglial cells cultivated on fibronectin. About 55% of the total APP amount was found in conditioned medium, 35% represented cellular immature APP isoforms, and about 10% represented mature APP isoforms. A higher amount of mature transmembrane APP together with a stronger suppression of APP secretion were observed using microglial cells cultivated on laminin and collagen. Whereas the cellular amount of APP from microglial cells cultivated on laminin already represented two-thirds of the total APP, microglial cells cultivated on collagen expressed more than three-fourths of their APP as cellular immature and mature APPs: 54% of total APP represented cellular immature APP, 28% represented cellular mature APP, and only 18% was detected in conditioned medium. After return of such nonadhesive cells to noncoated plastic culture dishes, secretion of APP increased rapidly, and the amount of mature transmembrane APP diminished (data not shown). Thus, under our experimental conditions, components of the extracellular matrix appear to be able to influence the APP biosynthesis and metabolism of the microglial cell line BV-2.

Accumulation of C-terminal APP Fragments in Nonadherent Microglial Cells

Since APP secretion is due to proteolytic cleavage within the C-terminal part of the extracellular domain, we have conducted Western blot analyses of microglial cells cultivated on the ECM molecules fibronectin (high APP secretion) and collagen (low APP secretion) in order to detect remaining C-terminal cell-associated fragments, as described by Estus et al.(51) and Golde et al.(52) . We used a monospecific anti-CT antibody. As shown in Fig. 4, APP-specific C-terminal fragments migrating at approximately 9-11 kDa were detected in both cell lysates. However, the amount of these C-terminal fragments differed. Interestingly, microglial cells with a relatively high level of APP secretion (fibronectin) showed a lowered level of C-terminal APP fragments, whereas C-terminal APP fragments accumulated in microglial cells with a suppressed APP secretion (collagen type I). This indicates an impaired degradation pathway in microglial cells growing in the presence of an unsuitable ECM substrate such as collagen.


Figure 4: Effect of different cultivation conditions on stability of APP-specific C-terminal fragments. Detergent extracts of BV-2 cells cultivated on fibronectin (fib) and collagen (coll) were analyzed for APP and APP-specific C-terminal fragments. Analysis of chloroform/methanol-precipitated protein was performed by SDS-PAGE using a Tris/Tricine gel containing 12.5% polyacrylamide followed by Western blot analysis using the monospecific anti-CT antibody. APP and APP-specific C-terminal fragments (CT frag) are indicated.



Treatment of Microglial Cells with Cytoskeleton-disrupting Agents

The observations reported above provided indirect evidence that the regulation of APP biogenesis and metabolism in microglial cells depends on cytoskeleton assembly. To further analyze the relationship between APP biogenesis and cytoskeletal organization, we cultivated microglial cells in the presence of the cytoskeleton-disrupting agents colchicine, nocodazole, and cytochalasins B and D. Colchicine and nocodazole are known to affect the assembly of microtubules; cytochalasins inhibit the polymerization of actin and might also have other disrupting effects on actin filament networks(53) .

Analysis was performed by chloroform/methanol precipitation followed by immunoblotting. The results are shown in Fig. 5. Using microglial cells cultivated on plastic substratum, only cytochalasin B was found to cause an accumulation of cellular mature APP and a concomitant increase in the amounts of C-terminal APP fragments (Fig. 5a). The secretion of APP was not significantly affected (Fig. 5b). Colchicine treatment had an enhancing effect on intracellular APP accumulation as well as on APP secretion, but no increase in C-terminal APP fragments was observed (Fig. 5b). Cytochalasin D did not alter the level of intracellular and extracellular APPs. Here, we observed a decrease in the level of APP-specific C-terminal fragments. Microglia treated with nocodazole showed low amounts of APP-specific C-terminal fragments and lower amounts of secretory APP.


Figure 5: Effect of cytoskeleton-disrupting agents on APP metabolism. BV-2 cells spread on plastic culture dishes were cultivated in the absence (control) or presence of different cytoskeleton-disrupting drugs: colchicine, cytochalasin B (cytochB), cytochalasin D (cytochD), and nocodazole. a, detection of transmembrane APP and C-terminal fragments. Detergent extracts of cells were precipitated by chloroform/methanol and separated by SDS-PAGE using a Tris/Tricine gel containing 12.5% polyacrylamide followed by Western blotting. Detection of transmembrane APP and C-terminal APP fragments was performed with the monospecific anti-CT antibody (dilution of 1:3000). b, detection of secreted APP. Conditioned medium of drug-treated microglial cells was subjected to immunoprecipitation with the anti-FdAPP serum. The immunoprecipitates were analyzed by immunoblotting. Detection of APP was performed with the monoclonal antibody 22C11 (dilution of 1:10,000).



From these data, we conclude that the accumulation of cellular mature transmembrane APP and the simultaneous increase in APP-specific C-terminal fragments predominantly depend on organization of the actin network. Since secretion of APP was not significantly decreased by treatment with agents disrupting the microglial cytoskeleton, we suggest that the intracellular accumulation of APP and APP fragments is not necessarily strongly coupled to the regulation of APP secretion.


DISCUSSION

Microglia play an important role during immunological processes, ontogenesis, and regeneration in the central nervous system(35, 36, 54, 55) . During such processes, interactions with surrounding cells and environment are necessary for functional activity. Changes in functional activity of microglia are often associated with morphological transformation(64) . A possible relevance of extracellular matrix proteins, particularly of fibronectin and laminin, to microglial differentiation and plasticity has been reported (42) .

The purpose of this investigation was to determine whether phenotypic alterations of microglial cells induced by extracellular matrix molecules are associated with changes in the APP biogenesis of microglial cells. The major finding of our studies is that changes in the adhesive state of microglia induced by components found in ECM may be significantly correlated with a specific APP metabolic behavior. Cell adhesion induced by substrates like fibronectin and polylysine predominantly leads to the secretion of APP. When the adherence of microglial cells to a surface was impaired by substrates like laminin and collagen, a significant down-regulation of APP secretion took place. At the same time, cellular mature transmembrane APP was markedly increased. These observations suggest that ECM influences the APP biogenesis and metabolism of the microglial cell line BV-2. The absence of a suitable substratum might support the formation of membrane junctional complexes between apposing cells.

As we have already suggested in a paper on immunocompetent cells(8) , transmembrane APP could serve directly as a cell adhesion molecule, while secreted APP might be involved in the regulation of cell interactions by generating intracellular signals via a yet undefined cell-surface receptor. These different cell interaction activities might be influenced by components of the extracellular matrix. It is of interest to know that neuronal re/degeneration of the brain is also associated with the expression of extracellular matrix proteins like fibronectin and laminin(40, 41) .

In injured brain, expression of a microglia-adhesive matrix molecule such as fibronectin might enhance the adherence of microglia, subsequently leading to an increased APP secretion. This may be an important signal for the recruitment of further microglia and part of the following regeneration or restoration process. Expression of low amounts of ECM proteins with anti-adhesive properties like laminin and collagen might diminish initial target cell binding of microglia and facilitate morphological transformations via soluble differentiation factors into an activated amoeboid phenotype. In contrast, high expression of laminin or collagen may abolish target cell interaction, thus leading to a deactivation of microglia. This would be in line with nerve regeneration being terminated. APP secretion would be suppressed, while an increased amount of mature transmembrane APP would be necessary for further microglial movements. However, since cell adhesion to ECM differs from cell type to cell type, this hypothetical reaction mechanism is strongly cell type-specific.

In vitro, isolated APP shows a relatively high binding affinity for collagen and laminin(17, 18, 56) . In vivo, microglia were predominantly nonadhesive when cultured on laminin and collagen, although high amounts of transmembranous APP were detectable. Thus, a possible in vivo binding of APP to collagen or laminin does not directly lead to cell adhesion, but may depend on the interaction with additional factors.

On the basis of immunohistochemical data, it has already been demonstrated that microglial cells are able to accumulate APP and APP fragments including betaA4-proteins(57, 58, 59) . However, the origin of these APP-related proteins remained unclear. We present evidence that intracellular APP metabolism and degradation occur in a cytoskeleton-dependent fashion. An association of APP with the cytoskeleton has also been shown by Refolo et al.(60) . Extracellular and intracellular matrices (cytoskeleton) are dynamically coupled through cell-surface receptors. ECM molecules may convey regulatory information through binding interactions with cytoskeletal proteins(33) . Loss of cell structure might be a consequence of failure of these interactions. We were able to demonstrate that actin microfilament disorganization gives rise to accumulation of both transmembrane APP and APP-specific C-terminal fragments. This might be due to an inhibition of distinct intracellular protein degradation mechanisms in microglial cells. We were not able to detect soluble intracellular or extracellular betaA4-proteins in the investigated murine microglial cell line BV-2. This might be ascribed to the origin of the investigated cell line BV-2, which was derived from murine microglia. In contrast to humans and several other higher mammals, mice and rats do not develop betaA4 depositions, pointing to differences in the APP metabolism of rodents and humans(61, 62) .

Since APP secretion was not impaired by treatment of microglial cells with cytoskeleton-disrupting agents, we conclude that APP secretion and intracellular APP metabolism may be regulated via different signals initiated by ECM-cell or cell-cell interactions. Direct interaction of mature transmembranous APP with components of the extracellular matrix like collagen and laminin might influence APP secretion by modulation of APP conformation. On the other hand, interactions of ECM molecules with their corresponding receptors may cause rearrangement of the cytoskeletal network (mentioned above) and, in addition, may activate an intracellular cascade of chemical signal pathways leading to changes in gene expression, e.g. expression of the APP gene and the APP secretase gene(33) .

In conclusion, our observations might provide a clue to the role of microglia in the pathology of Alzheimer's disease since microglia are closely associated with certain amyloid plaque deposits in the brain of Alzheimer patients(41, 42, 63) . Although the precise function of microglia in amyloidogenesis is unknown, our data indicate that microglia can play a key role in initial events of amyloidogenesis in Alzheimer's disease. Intracellular accumulation of APP and also of amyloidogenic APP fragments in response to physiological changes may be relevant to amyloid depositions. It is an intriguing hypothesis that microglia initiate amyloid plaque formation by an accumulation of amyloidogenic APP fragments. This process might be triggered by the expression of certain components of the extracellular matrix upon brain injury.


FOOTNOTES

*
This work was supported by Deutsche Forschungsgemeinschaft Grants SFB 317 and SFB 258 and by grants from the Bundesminister für Forschung und Technologie of Germany, the Metropolitan Life Foundation, the Fonds der Chemischen Industrie of Germany, the Forschungsschwerpunkt Baden-Württemberg (to K. B.), the National Health and Medical Research Council of Australia, the Victorian Health Promotion Foundation, and the Aluminium Development Corporation of Australia (to C. L. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

()
To whom correspondence should be addressed: Research Laboratories of Schering AG, D-13342 Berlin, Germany. Tel.: 49-30-4682177; Fax: 49-30-4691-6738.

(^1)
The abbreviations used are: APP, betaA4-amyloid precursor protein; L-APP, leukocyte-derived APP; APLP, APP-like protein; ECM, extracellular matrix; Tricine; N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis; CT, C-terminal.


ACKNOWLEDGEMENTS

We thank V. Bocchini for providing the microglial cell line BV-2 and K. H. Scheit for providing the APLP2 cDNA.


REFERENCES

  1. Müller-Hill, B., and Beyreuther, K. (1989) Annu. Rev. Biochem. 58, 287-307 [CrossRef][Medline] [Order article via Infotrieve]
  2. Selkoe, D. J. (1993) Trends Neurosci. 16, 403-409 [CrossRef][Medline] [Order article via Infotrieve]
  3. Kang, J., Lemaire, H. G., Unterbeck, A., Salbaum, J. M., Masters, C. L., Grzeschik, K. H., Multhaup, G., Beyreuther, K., and Müller-Hill, B. (1987) Nature 325, 733-736 [CrossRef][Medline] [Order article via Infotrieve]
  4. Ponte, P., Gonzalez, D. P., Schilling, J., Miller, J., Hsu, D., Greenberg, B., Davis, K., Wallace, W., Lieberburg, I., and Fuller, F. (1988) Nature 331, 525-527 [CrossRef][Medline] [Order article via Infotrieve]
  5. Kitaguchi, N., Takahashi, Y., Tokushima, Y., Shiojiri, S., and Ito, H. (1988) Nature 331, 530-532 [CrossRef][Medline] [Order article via Infotrieve]
  6. Tanzi, R. E., McClatchey, A. I., Lamperti, E. D., Villa, K. L., Gusella, J. F., and Neve, R. L. (1988) Nature 331, 528-530 [CrossRef][Medline] [Order article via Infotrieve]
  7. König, G., Mönning, U., Czech, C., Prior, R., Banati, R., Schreiter-Gasser, U., Bauer, J., Masters, C. L., and Beyreuther, K. (1992) J. Biol. Chem. 267, 10804-10809 [Abstract/Free Full Text]
  8. Mönning, U., König, G., Banati, R. B., Mechler, H., Czech, C., Gehrmann, J., Schreiter-Gasser, U., Masters, C. L., and Beyreuther, K. (1992) J. Biol. Chem. 267, 23950-23956 [Abstract/Free Full Text]
  9. Banati, R. B., Gehrmann, J., Czech, C., Mönning, U., Jones, L. L., König, G., Beyreuther, K., and Kreutzberg, G. W. (1993) Glia 9, 199-210 [Medline] [Order article via Infotrieve]
  10. Sandbrink, R., Masters, C. L., and Beyreuther, K. (1994) J. Biol. Chem. 269, 1510-1517 [Abstract/Free Full Text]
  11. Esch, F. S., Keim, P. S., Beattie, E. C., Blacher, R. W., Culwell, A. R., Oltersdorf, T., McClure, D., and Ward, P. J. (1990) Science 248, 1122-1124 [Medline] [Order article via Infotrieve]
  12. Sisodia, S. S., Koo, E. H., Beyreuther, K., Unterbeck, A., and Price, D. L. (1990) Science 248, 492-495 [Medline] [Order article via Infotrieve]
  13. Haass, C., Schlossmacher, M. G., Hung, A. Y., Vigo, P. C., Mellon, A., Ostaszewski, B. L., Lieberburg, I., Koo, E. H., Schenk, D., Teplow, D. B., and Selkoe, D. J. (1992) Nature 359, 322-325 [CrossRef][Medline] [Order article via Infotrieve]
  14. Seubert, P., Vigo, P. C., Esch, F., Lee, M., Dovey, H., Davis, D., Sinha, D., Schlossmacher, M., Whaley, J., Swindlehurst, C., McCormack, R., Wolfert, R., Selkoe, D., Lieberburg, I., and Schenk, D. (1992) Nature 359, 325-327 [CrossRef][Medline] [Order article via Infotrieve]
  15. Shoji, M., Golde, T. E., Ghiso, J., Cheung, T. T., Estus, S., Shaffer, L. M., Cai, X. D., McKay, D. M., Tintner, R., Frangione, B., and Younkin, S.G. (1992) Science 258, 126-129 [Medline] [Order article via Infotrieve]
  16. Klier, F. G., Cole, G., Stallcup, W., and Schubert, D. (1990) Brain. Res. 515, 336-342 [Medline] [Order article via Infotrieve]
  17. Narindrasorasak, S., Lowery, D., Gonzalez-DeWhitt, P., Poorman, R. A., Greenberg, B., and Kisilevsky, R. (1991) J. Biol. Chem. 266, 12878-12883 [Abstract/Free Full Text]
  18. Narindrasorasak, S., Lowery, D. E., Altman, R. A., Gonzalez, D. P., Greenberg, B. D., and Kisilevsky, R. (1992) Lab. Invest. 67, 643-652 [Medline] [Order article via Infotrieve]
  19. Breen, K. C. (1992) Mol. Chem. Neuropathol. 16, 109-121 [Medline] [Order article via Infotrieve]
  20. Ninomiya, H., Roch, J. M., Sundsmo, M. P., Otero, D. A., and Saitoh, T. (1993) J. Cell Biol. 121, 879-886 [Abstract]
  21. Oltersdorf, T., Fritz, L. C., Schenk, D. B., Lieberburg, I., Johnson, W. K., Beattie, E. C., Ward, P. J., Blacher, R. W., Dovey, H. F., and Sinha, S. (1989) Nature 341, 144-147 [CrossRef][Medline] [Order article via Infotrieve]
  22. Van Nostrand, W., Wagner, S. L., Suzuki, M., Choi, B. H., Farrow, J. S., Geddes, J. W., Cotman, C. W., and Cunningham, D. D. (1989) Nature 341, 546-549 [CrossRef][Medline] [Order article via Infotrieve]
  23. Van Nostrand, W., Schmaier, H., Farrow, J. S., and Cunningham, D. D. (1990) Science 248, 745-748 [Medline] [Order article via Infotrieve]
  24. Smith, R. P., Higuchi, D. A., and Broze, G. J. (1990) Science 248, 1126-1128 [Medline] [Order article via Infotrieve]
  25. Bush, A. I., Martins, R. N., Rumble, B., Moir, R., Fuller, S., Milward, E., Currie, J., Ames, D., Weidemann, A., Fischer, P., Multhaup, G., Beyreuther, K., and Masters, C. L. (1990) J. Biol. Chem. 265, 15977-15983 [Abstract/Free Full Text]
  26. Shivers, B. D., Hilbich, C., Multhaup, G., Salbaum, M., Beyreuther, K., and Seeburg, P. H. (1988) EMBO J. 7, 1365-1370 [Abstract]
  27. Schubert, D., Jin, L.-W., Saitoh, T., and Cole, G. (1989) Neuron 3, 689-694 [Medline] [Order article via Infotrieve]
  28. Schubert, W., Prior, R., Weidemann, A., Dircksen, H., Multhaup, G., Masters, C. L., and Beyreuther, K. (1991) Brain Res. 563, 184-194 [CrossRef][Medline] [Order article via Infotrieve]
  29. Breen, K. C., Bruce, M., and Anderton, B. H. (1991) J. Neurosci. Res. 28, 90-100 [Medline] [Order article via Infotrieve]
  30. König, G., Masters, C. L., and Beyreuther, K. (1990) FEBS Lett. 269, 305-310 [CrossRef][Medline] [Order article via Infotrieve]
  31. Milward, E. A., Papadopoulos, R., Fuller, S. J., Moir, R. D., Small, D., Beyreuther, K., and Masters, C. L. (1992) Neuron 9, 129-137 [Medline] [Order article via Infotrieve]
  32. Nitsch, R. M., Slack, B. E., Wurtman, R. J., and Growdon, J. H. (1992) Science 258, 304-307 [Medline] [Order article via Infotrieve]
  33. Lin, C. Q., and Bissell, M. J. (1993) FASEB J. 7, 737-743 [Abstract/Free Full Text]
  34. Juliano, R. L., and Haskill, S. (1993) J. Cell Biol. 120, 577-585 [Medline] [Order article via Infotrieve]
  35. Perry, V. H., and Gordon, S. (1988) Trends Neurosci. 11, 273-279 [CrossRef][Medline] [Order article via Infotrieve]
  36. Streit, W. J., Graeber, M. B., and Kreutzberg, G. W. (1988) Glia 1, 301-307 [Medline] [Order article via Infotrieve]
  37. Graeber, M. B., and Streit, W. J. (1990) Brain Pathol. 1, 2-5 [Medline] [Order article via Infotrieve]
  38. del Rio-Hortega, P. (1932) in Cytology and Cellular Pathology of the Nervous System (Penfield, W., ed) pp. 481-543, de Hoever Inc., New York
  39. Perry, V. H., and Gordon, S. (1991) Int. Rev. Cytol. 125, 203-244 [Medline] [Order article via Infotrieve]
  40. Liesi, P., Kaakkola, S., Dahl, D., and Vaheri, A. (1984) EMBO J. 3, 683-686 [Abstract]
  41. Lefcort, F., Venstrom, K., McDonald, J. A., and Reichardt, L. F. (1992) Development (Cambr.) 116, 767-782 [Abstract/Free Full Text]
  42. Chamak, B., and Mallat, M. (1991) Neuroscience 45, 513-527 [CrossRef][Medline] [Order article via Infotrieve]
  43. Haga, S., Akai, K., and Ishii, T. (1989) Acta Neuropathol. 77, 569-575 [Medline] [Order article via Infotrieve]
  44. Wisniewski, H. M., Wegiel, J., Wang, K. C., Kujawa, M., and Lach, B. (1989) Can. J. Neurol. Sci. 16, 535-542 [Medline] [Order article via Infotrieve]
  45. Blasi, E., Barluzzi, R., Bocchini, V., Mazzolla, R., and Bistoni, F. (1990) J. Neuroimmunol. 27, 229-237 [CrossRef][Medline] [Order article via Infotrieve]
  46. Weidemann, A., König, G., Bunke, D., Fischer, P., Salbaum, J. M., Masters, C. L., and Beyreuther, K. (1989) Cell 57, 115-126 [Medline] [Order article via Infotrieve]
  47. Mönning, U., Sandbrink, R., Banati, R. B., Masters, C. L., and Beyreuther, K. (1994) FEBS Lett. 342, 267-272 [CrossRef][Medline] [Order article via Infotrieve]
  48. Deleted in proof
  49. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  50. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Ehrlich, H. A. (1988) Science 239, 487-491 [Medline] [Order article via Infotrieve]
  51. Estus, S., Golde, T. E., Kunishita, T., Blades, D., Lowery, D., Eisen, M., Usiak, M., Qu, X. M., Tabira, T., Greenberg, B. D., and Younkin, S. G. (1992) Science 255, 726-728 [Medline] [Order article via Infotrieve]
  52. Golde, T. E., Estus, S., Younkin, L. H., Selkoe, D. J., and Younkin, S. G. (1992) Science 255, 728-730 [Medline] [Order article via Infotrieve]
  53. Forscher, P., and Smith, S. J. (1988) J. Cell Biol. 107, 1505-1516 [Abstract]
  54. Gehrmann, J., Gold, R., Linnington, C., Lannes-Viera, J., Wekerle, H., and Kreutzberg, G. W. (1992) Lab. Invest. 67, 100-113 [Medline] [Order article via Infotrieve]
  55. Guilian, D., and Ingemann, J. E. (1988) J. Neurosci. 8, 4707-4717 [Abstract]
  56. Multhaup, G., Bush, A., Pollwein, P., Masters, C. L., and Beyreuther, K. (1992) J. Protein Chem. 11, 298-299
  57. Haass, C., Hung, A. Y., and Selkoe, D. J. (1991) J. Neurosci. 11, 3783-3793 [Abstract]
  58. Shigematsu, K., and McGeer, P. L. (1992) Brain Res. 593, 117-123 [Medline] [Order article via Infotrieve]
  59. Frackowiak, J., Wisniewski, H. M., Wegiel, J., Merz, G. S., Iqbal, K., and Wang, K. C. (1992) Acta Neuropathol. 84, 225-233 [Medline] [Order article via Infotrieve]
  60. Refolo, L. M., Wittenberg, I. S., Friedrich, V. J., and Robakis, N. K. (1991) J. Neurosci. 11, 3888-3897 [Abstract]
  61. Hilbich, C., Kisters-Woike, B., Reed, J., Masters, C. L., and Beyreuther, K. (1991) Eur. J. Biochem. 201, 61-69 [Abstract]
  62. Fraser, P. E., Nguyen, J. T., Inouye, H., Surewicz, W. K., Selkoe, D. J., Podlisny, M. D., and Kirschner, A. (1992) Biochemistry 31, 10716-10723 [Medline] [Order article via Infotrieve]
  63. Perlmutter, L. S., Barron, E., and Chui, H. C. (1990) Neurosci. Lett. 119, 32-36 [CrossRef][Medline] [Order article via Infotrieve]
  64. Ling, E.-A., and Wong, W.-C. (1993) Glia 7, 9-18 [Medline] [Order article via Infotrieve]
  65. Wasco, W., Gurubhagavatula, S., Paradis, M. D., Romano, D. M., Sisodia, S. S., Hyman, B. T., Neve, R. L., and Tanzi, R. E. (1993) Nature Genetics 5, 95-100 [Medline] [Order article via Infotrieve]
  66. Sprecher, C. A., Grant, F. J., Grimm, G., O'Hara, P. J., Norris, F., Norris, K., and Foster, D. C. (1993) Biochemistry. 32, 4481-4486 [Medline] [Order article via Infotrieve]

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