(Received for publication, February 7, 1994; and in revised form, October 27, 1994)
From the
During axotomy studies, we discovered that the A4-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.
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 A4-peptide that is generated by
proteolytic cleavage from a parent amyloid protein precursor (APP). (
)
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 A4-peptides can be released in the
extracellular milieu upon proteolytic breakdown of transmembrane APP.
The
A4-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
A4 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/A4 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.
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.
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.
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 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).
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.
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.
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.
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
A4-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
A4-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
A4
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.