Degradation of Amyloid beta -Protein by a Metalloprotease Secreted by Microglia and Other Neural and Non-neural Cells*

(Received for publication, December 4, 1996, and in revised form, December 27, 1996)

Wei Qiao Qiu , Zhen Ye , Dora Kholodenko Dagger , Peter Seubert Dagger and Dennis J. Selkoe §

From the Center for Neurologic Diseases, Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts 02115 and Dagger  Athena Neurosciences, Inc., South San Francisco, California 94080

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Amyloid beta -protein (Abeta ) is the major component of neuritic (amyloid) plaques in Alzheimer's disease, and its deposition is an early and constant event in the complex pathogenetic cascade of the disease. Although many studies have focused on the biosynthetic processing of the beta -amyloid precursor protein and on the production and polymerization of Abeta , understanding the degradation and clearance of Abeta has received very little attention. By incubating the conditioned medium of metabolically labeled Abeta -secreting cells with media of various cultured cell lines, we observed a time-dependent decrease in the amount of Abeta in the mixed media. The factor principally responsible for this decrease was a secreted metalloprotease released by both neural and non-neural cells. Among the cells examined, the microglial cell line, BV-2, produced the most Abeta -degrading activity. The protease was completely blocked by the metalloprotease inhibitor, 1,10-phenanthroline, and partially inhibited by EDTA, whereas inhibitors of other protease classes produced little or no inhibition. Substrate analysis suggests that the enzyme was a non-matrix metalloprotease. The protease cleaved both Abeta 1-40 and Abeta 1-42 peptides secreted by beta -amyloid precursor protein-transfected cells but failed to degrade low molecular weight oligomers of Abeta that form in the culture medium. Lipopolysaccharide, a stimulator of macrophages/microglia, activated BV-2 cells to increase their Abeta -degrading metalloprotease activity. We conclude that secreted Abeta 1-40 and Abeta 1-42 peptides are constitutively degraded by a metalloprotease released by microglia and other neural cells, providing a potential mechanism for the clearance of Abeta in brain tissue.


INTRODUCTION

The defining pathological features of Alzheimer's disease (AD)1 are extracellular deposits of amyloid beta -proteins (Abeta ) that form senile plaques and amyloid angiopathy and intraneuronal deposits of modified tau proteins that form neurofibrillary tangles. Abeta are 40-43-amino acid proteolytic fragments generated by unidentified proteases from the transmembrane glycoprotein, beta -amyloid precursor protein (beta APP) (1, 2). The gene encoding beta APP is on human chromosome 21q, and missense mutations in and around the Abeta coding region of this gene are a rare cause of familial AD (FAD). Moreover, trisomy 21 (Down's syndrome) is characterized by overexpression of beta APP due to increased gene dosage, resulting in very early Abeta deposition followed by the gradual development of the classical neuropathological lesions of AD (3, 4). Recently, mutations in the presenilin 1 and 2 genes, which cause severe early onset FAD (5-7), have been shown to selectively increase the production and cerebral deposition of the highly amyloidogenic 42-residue form of Abeta (8, 9). These and other findings provide strong evidence that disordered beta APP metabolism can increase the production of Abeta peptides, particularly the Abeta 1-42 peptide, thereby initiating amyloid plaque formation and the pathological cascade of AD. In support of this model, transgenic mice overexpressing the V717F mutation or K670N/M671L mutation of beta APP progressively develop Abeta deposits and plaque-associated neuritic, microglial, and astrocytic pathology with age and may even show concomitant memory impairment (10-12).

Much attention has been focused on the secretory and endocytic processing of beta APP by cells in order to understand how Abeta is generated normally and in AD. In contrast, little is known about how Abeta , once secreted, is degraded and cleared from tissues. At present, only the relatively rare forms of FAD linked to the beta APP or presenilin genes are thought to involve overproduction of Abeta . The excessive cerebral accumulation of Abeta that occurs in all other cases of the disease could be explained in part by a decrease in the ability of the brain to degrade and clear Abeta . If specific Abeta -degrading proteases can be shown to be released by neural cells, changes in the structure or activity of such proteases could be sought in as yet unexplained forms of FAD, and their up-regulation could represent a therapeutic approach to AD in general.

Here, we have screened certain neural and non-neural cell lines to ascertain whether they constitutively release proteases capable of degrading the Abeta peptides naturally secreted by cells. We have identified a secreted metalloprotease activity that efficiently degrades endogenous Abeta into fragments under cell culture conditions. Among the neural cell lines tested, a microglial line, BV-2, secretes the active protease robustly. Substrate analysis suggests that it is a non-matrix metalloprotease. Its production or activity is up-regulated by activating BV-2 cells. Interestingly, the metalloprotease is far more effective in degrading secreted Abeta 40 and Abeta 42 monomers than Abeta oligomers formed in cell culture. These findings have implications for the normal and pathological clearance of Abeta in brain.


EXPERIMENTAL PROCEDURES

Cell Culture

Chinese hamster ovary (CHO) cells stably transfected with beta APP770 cDNA containing the Val right-arrow Phe mutation at residue 717 (7PA2 cells) (13) were routinely cultured in Dulbecco's modified Eagle's medium, 10% fetal bovine serum (FBS) with G418 (200 µg/ml). Untransfected CHO cells, monkey kidney COS cells, and the human neuroblastoma cell lines, M17 and SY5Y, were grown in Dulbecco's modified Eagle's medium, 10% FBS. All these cells were passaged by adding 3 ml of 50 mM EDTA, immediately aspirating it off, and incubating the cells at 37 °C for 2 min followed by washing with Hanks' balanced salt solution (Life Technologies, Inc.). The cells were passaged at 1:10 dilution. Mouse microglial cells, BV-2, were cultured in RPMI 1640, 10% FBS (14). Because they were semisuspended, BV-2 cells were shaken to be lifted during passage.

Assays of Abeta -degrading Activity

To obtain conditioned media (CM) containing Abeta -degrading activity, different cultured cells were washed 3 times with serum-free N2 medium (N2 supplement (Life Technologies, Inc.), 1% ovalbumin, 1 mM pyruvate in MEM) (N2). N2 was added, and cells were conditioned at 37 °C for various times. CM were collected and centrifuged at 3,000 × g for 30 min to remove cells. Some CM were concentrated 10 times in a Centricon 30 (Amicon) filter. To characterize Abeta -degrading activity, confluent monolayers of 7PA2 beta APP-transfected CHO cells in 10-cm dishes were preincubated for 30 min in methionine-free medium and labeled for 4 h with 300 µCi of [35S]methionine. The labeled media were collected, combined, and centrifuged at 3,000 × g for 30 min. An amount of labeled medium (3 ml) was mixed with an equal amount of cell conditioned medium from the various cells being tested or with unconditioned N2 medium as a control, and the mixtures were incubated at 37 °C for 16 h. The amount of labeled Abeta remaining in each sample was assessed by immunoprecipitation with the high affinity Abeta antibody, R1282 (4). This antibody was generated to synthetic Abeta 1-40 peptide and characterized similarly to our Abeta antibody, R1280, used in our previous study (15).

Abeta degradation in cultures was also quantified by incubating 7 µl of conditioned or unconditioned medium with 125I-labeled Abeta 1-40 (3.3 × 104 cpm) (kind gift of Dr. John Maggio) (15) in 14 µl of reaction buffer (187 mM NaCl, 0.02 M NaH2PO4, 10 mM Tris-HCl, pH 7.5) at 37 °C for 16 h. After quenching the reaction with 7 µl of glacial acetic acid and 5 µl of 0.1% pyronin Y, the sample was analyzed by acid-urea-PAGE (15) and autoradiography.

Protease inhibition assays were conducted by adding different protease inhibitors at the indicated concentrations to the mixtures before incubating at 37 °C for 16 h. All inhibitors shown in Table I were purchased from Sigma.

Table I.

Inhibition profile of Abeta -degrading enzyme in the CHO conditioned medium

Amounts of exogenously added Abeta 1-40 remaining undegraded after incubation in the CHO conditioned medium in the presence of different protease inhibitors were determined using a sandwich ELISA assay. The amount of Abeta 1-40 after incubation in the unconditioned medium, N2, was 4.10 ng/ml, while the amount of Abeta 1-40 remained in the CHO conditioned medium without the protease inhibitors is 0.37 ng/ml. Therefore, the Abeta -degrading activity in the original CHO conditioned medium was set as 0% inhibition; recovery of Abeta 1-40 level to that in N2 medium represents 100% inhibition. TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; DFP, diisopropyl phosphofluoridate.
Inhibitor Protease class Concentration Concentration of Abeta Inhibition of degradation of Abeta

ng/ml %
E64 Cysteine 10 µM 0.38 <0.5
Pepstatin A Aspartic 1 µM 0.46 2
EDTA Metallo 5 mM 0.63 7
1,10-Phenanthroline Metallo 10 mM 4.41 100
TPCK Serine 100 µM 0.34 0
Aprotinin Serine 100 µM 0.43 2
Elastatinal Serine 100 µM 0.42 1
Pefabloc Serine 1 mM 0.47 3
DFP Serine 100 µM 0.33 0

ELISA

Cold 4-h conditioned medium of 7PA2 cells was mixed with plain N2 or with CM of CHO or BV-2 cells. After incubating at 37 °C for 16 h, total Abeta or specific Abeta 1-42 remaining in each sample was quantified by highly specific sandwich ELISA assays described previously (16). Two different ELISA assays were performed using distinct capture antibodies: 266 (raised against residues 13-28 of Abeta ), for total Abeta or 21F12 (raised against residues 33-42 of Abeta ), for Abeta peptides ending specifically at residue 42. The reporter antibody was 3D6 (to residues 1-5 of Abeta ) in both assays. The ratio of Abeta 1-42 over total Abeta in each sample was calculated.

LPS Treatment

Bacterial lipopolysaccharide (LPS) (Escherichia coli serotype O111:B4) was purchased from Calbiochem. Cells were either left unstimulated or stimulated with LPS (10 µg/ml) in culture medium containing 10% FBS at 37 °C for various times. Conditioned media were collected and centrifuged before assaying Abeta -degrading activity as described above.


RESULTS

Decrease of Abeta Is Mediated by the Conditioned Media of Non-neural and Neural Cells

We examined several non-neural and neural cell lines for the secretion of an Abeta -degrading protease activity: COS monkey kidney cells; Chinese hamster ovary (CHO) cells; the human neuroblastoma cell lines, M17 and SY5Y; and the murine microglial line, BV-2. All cultures were passaged by brief incubation with EDTA rather than trypsin, in order to avoid the formation of a serine protease/alpha 2-macroglobulin complex in the medium, which we recently showed can occur during tryptic passage of cells and degrade secreted Abeta (15). To search for Abeta -degrading activity, confluent cultures were washed and changed to the serum-free medium, N2, for further cultivation. After conditioning for 24 h, the media were collected and centrifuged to remove floating cells. CM from certain cell lines (CHO, COS, M17, and SY5Y) were concentrated 10-fold by Centricon filtration before assaying for proteolytic activity. To detect degradation of endogenously secreted Abeta , CHO cells stably transfected with beta APP770 cDNA containing the V717F mutation (7PA2 cells; Ref. 13) were labeled with [35S]Met in serum-free medium for 4 h, and the resultant medium (containing abundant labeled Abeta and p3 (17) peptides) was incubated for 16 h at 37 °C with either unconditioned N2 medium or the CM of the aforementioned cell lines. Because no cells are present during the incubation, any loss of Abeta and p3 cannot be due to internalization of the peptides or any other cell-associated function. By immunoprecipitating the mixed media with a high titer Abeta antibody (R1282) and performing gel fluorography, we observed variable declines in the amounts of Abeta and p3 compared to those in plain N2 medium (Fig. 1). The amounts of total secreted proteins and APPs in the incubated CM changed little or not at all (data not shown). Among the cell lines we examined, CHO and BV-2 CM produced the largest loss of Abeta after incubation (Fig. 1).


Fig. 1. Decrease of secreted Abeta is mediated by the conditioned media of various cell lines. A, CHO cells stably transfected with an APP770 gene containing V717F mutation (7PA2) were washed and labeled with [35S]methionine for 4 h in serum-free medium. Equal amounts of the labeled supernatant were incubated with the 10 × concentrated unconditioned N2 medium (lane 1) or 10 × concentrated CM of CHO (lane 2), or COS (lane 3) cells at 37 °C for 16 h. The incubated media were immunoprecipitated with the Abeta antibody, R1282, and electrophoresed on Tris-Tricine SDS-PAGE gels, followed by autoradiography. Molecular size markers (kDa) are indicated. B, aliquots of the labeled 7PA2 supernatant were incubated 1:1 with 10 × concentrated unconditioned N2 medium (lane 1) or 10 × concentrated CM of CHO, COS M17, and SY5Y cells at 37 °C for 16 h. The incubated media were immunoprecipitated as described in A. C, equal amounts of the labeled 7PA2 CHO CM were incubated with unconditioned medium (lane 1), CHO CM (lane 2), or BV-2 CM (lane 3) at 37 °C for 16 h, followed by characterization as in A.
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To obtain quantitative information about Abeta degradation by these two cell types, we assayed the CM obtained from cultures of known cell densities grown in a standard volume of medium. The amount of Abeta remaining in the CM of 2.5 × 107 BV-2 cells was consistently less than that in CM of this number of CHO cells (Fig. 2A), indicating that the microglial cell line secreted more Abeta -degrading activity. Conditioning these cell lines for increasing intervals revealed that the confluent CHO cells produced little or no Abeta -degrading activity in the first 6 h and generated maximal activity by 24 h (Fig. 2B). In the case of BV-2 cells (1 × 107), conditioning for 2 h already produced substantial activity, further suggesting that the microglial cell CM caused the most degradation among the lines we characterized.


Fig. 2. Comparison of the decrease in secreted Abeta after incubation in CHO or BV-2 conditioned medium. A, CM were collected from 2.7 × 107 CHO cells (lane 2), 5 × 106 BV-2 cells (lane 3), 1 × 107 BV-2 cells (lane 4), and 2.5 × 107 BV-2 cells (lane 5), respectively. These media and unconditioned N2 medium (lane 1) were incubated with equal aliquots of [35S]methionine-labeled 7PA2 CHO medium at 37 °C for 16 h followed by immunoprecipitation as in Fig. 1. B, confluent CHO cells were grown in N2 medium at 37 °C for 2, 6, 16, or 24 h. These CM and unconditioned N2 medium (lane 1) were incubated with the labeled 7PA2 medium, followed by immunoprecipitation with R1282. C, BV-2 cells (1 × 107) were grown in N2 medium for 0.5, 1, 2, 6, 16, or 24 h. The CM and unconditioned N2 medium (lane 1) were incubated with labeled 7PA2 medium followed by R1282 immunoprecipitation.
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Abeta Degradation Is Mediated by a Secreted Metalloprotease in Both CHO and BV-2 Conditioned Media

To determine whether that the loss of Abeta in CM was caused by degradation by secreted protease(s), we examined the effects of several different protease inhibitors with an assay similar to that described above. Using concentrations known to cause maximal inhibition of other secreted proteases (18), various well characterized inhibitors were incubated for 24 h at 37 °C with either N2 medium or CHO CM in the presence of synthetic Abeta 1-40 peptide (40 ng/ml) as a substrate, followed by a sandwich ELISA to detect remaining intact Abeta . The inhibitor profile clearly demonstrated an Abeta -degrading metalloprotease in the CHO medium (Table I). The degradation of cell-secreted Abeta and p3 by both CHO and BV-2 media was essentially abolished by 1,10-phenanthroline, whereas EDTA generally produced less inhibition (Fig. 3). The different degrees of inhibition of the degradation of endogenous (Fig. 3) versus synthetic (Table I) Abeta by EDTA are unexplained, but may be due to different conformations of these two substrates, and we are conducting experiments to address this issue. Although phosphoramidon is also a metalloprotease inhibitor, it did not inhibit the degradation of either synthetic or endogenous Abeta in this system (Fig. 3). These results indicate that the major Abeta -degrading activity present in both CHO and BV-2 CM is a secreted metalloprotease. Another protease inhibitor, 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc), produced slight inhibition of Abeta degradation (Fig. 3), suggesting that a serine protease may also make a small contribution to Abeta proteolysis by CHO and BV-2 media.


Fig. 3. Degradation of Abeta by CHO or BV-2 conditioned media is blocked by certain metalloprotease inhibitors. Abeta secreted by 7PA2 CHO cells was analyzed as in Fig. 1 after incubating with unconditioned N2 medium (lane 1 of A and B), CM of CHO cells (lanes 2-7 in A) or CM of BV-2 cells (lanes 2-7 in B). The protease inhibitors E-64 (lane 3), EDTA (lane 4), 1,10-phenanthroline (lane 5), phosphoramidon (lane 6), or Pefabloc (lane 7) were added to the mixed media before incubation at 37 °C for 16 h. Abeta and p3 (arrows) were precipitated by R1282.
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To detect the products of the Abeta degradation, acid-urea-PAGE (15) was used to analyze 125I-labeled synthetic Abeta 1-40 after incubation in CHO or BV-2 CM for 24 h (Fig. 4). The amount of intact Abeta decreased markedly, and 3 peptide bands appeared that migrated differently from intact Abeta and were not produced by incubation in N2 medium alone. The production of these fragments was completely inhibited by 1,10-phenanthroline, confirming that Abeta is degraded by a secreted metalloprotease in both CHO and BV-2 CM and generates similar peptide products.


Fig. 4. Detection of products of Abeta degradation by CHO or BV-2 conditioned medium. 125I-Labeled synthetic Abeta 1-40 peptide was incubated 37 °C, for 16 h with either unconditioned N2 medium (lane 1) or CHO CM (lanes 2 and 3) or BV-2 CM (lanes 4 and 5). Proteolytic products formed in the absence (lanes 2 and 4) but not the presence (lanes 3 and 5) of 1,10-phenanthroline were resolved by 15% acid-urea-PAGE and stained with Coomassie Blue. Intact Abeta 1-40 and the Abeta cleavage products are indicated.
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Degradation of Abeta 40, Abeta 42 and Abeta Oligomers by the Metalloprotease

Because Abeta peptides ending at residue 42 (Abeta 1-42) are the initially deposited species in AD and normal aged brain tissue, we examined the substrate specificity of the metalloprotease on endogenous Abeta 1-40 versus Abeta 1-42 peptides. Confluent 7PA2 cells were washed and cultured in serum-free N2 medium for 4 h. Equal amounts of this CM were incubated with plain N2, CHO CM, or BV-2 CM for 24 h, followed by sandwich ELISAs measuring either total Abeta (including Abeta 1-40 and Abeta 1-42) or Abeta 1-42 alone. The amounts of total Abeta and Abeta 1-42 remaining in CHO and BV-2 media were decreased approximately 75% compared to those in N2 (Fig. 5, A and B). As a result, the ratios of Abeta 1-42 to total Abeta showed only a slight and insignificant increase (Fig. 5C).


Fig. 5. ELISA quantitation of degradation of secreted Abeta 40 and Abeta 42 peptides by the metalloprotease in CHO and BV-2 conditioned media. Confluent 7PA2 CHO cells were incubated in serum-free N2 medium at 37 °C for 4 h. The CM was then incubated with N2 alone, CHO CM, or BV-2 CM for 16 h. The residual amounts of total Abeta peptides (A) and of Abeta 1-42 (B) were analyzed by sandwich ELISA. The ratio of Abeta 1-42 over the total Abeta in each condition was calculated (C).
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We also characterized the specificity of the metalloprotease in the 24-h conditioned media of CHO and BV-2 cells for Abeta -related species found in the culture medium of our beta APP-expressing 7PA2 cells. Abeta was substantially decreased by 4 h of incubation, whereas p3 showed relative stability for the first 8 h of incubation (Fig. 6). Importantly, the endogenous 6-12- kDa oligomeric Abeta species that we previously documented in the CM of these V717F beta APP-transfected CHO cells (13) were completely resistant to proteolysis (Fig. 6).


Fig. 6. p3 and low molecular weight Abeta oligomers are relatively resistant to degradation by the metalloprotease. Overnight labeled 7PA2 medium was incubated with unconditioned N2 medium (lanes 1, 4, 7, 10, and 13), CHO CM (lanes 2, 5, 8, 11, and 14) or BV-2 CM (lanes 3, 6, 9, 12, and 15) at 37 °C for the indicated reaction times. Labeled Abeta and p3 (arrows) were then precipitated with R1282 and analyzed as in Fig. 1. Labeled oligomeric Abeta bands (8, 12, and 16 kDa), which were fully characterized previously (13), are shown above the monomeric Abeta .
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Regulation of the Secreted Metalloprotease

During the course of these studies, we noticed that high Abeta -degrading activity in CM (>90% decrease of Abeta compared to that in N2) occurred when cells were conditioned in the absence of serum. In contrast, only a 10-15% decrease in Abeta occurred when 10% FBS was present during conditioning (data not shown). To clarify the role of serum in the production and activity of the Abeta -degrading protease, we conditioned CHO cells in N2 medium containing increasing concentrations of FBS for 24 h. These CM were then incubated with labeled CHO 7PA2 medium at 37 °C for 16 h, followed by the Abeta immunoprecipitation assay described above. As a control, 10% FBS was added to a sample of medium conditioned without serum just prior to the incubation. CHO CM containing 1% FBS degraded Abeta as much as CHO CM without FBS, whereas the amount of Abeta in CHO CM containing 10% FBS appeared virtually unchanged from that in plain N2 (Fig. 7). Increasing the serum concentration between 1% and 10% led to a graded decrease in the amount of Abeta -degrading activity (Fig. 7). Using the ELISA assay, we were similarly able to detect only a 10-20% decrease in Abeta levels in medium conditioned in 10% FBS (data not shown). These results indicate that increasing the percentage of serum during conditioning results in a corresponding decrease in Abeta proteolysis. On the other hand, adding 10% FBS solely during the incubation period did not decrease the strong Abeta -degrading activity generated by CHO cells conditioned without serum. Serum also decreased the metalloprotease activity released from BV-2 cells but to a lesser degree than CHO cells (Fig. 8B). Thus, serum appears to decrease the cellular production of the Abeta -degrading metalloprotease or increase the secretion of a metalloprotease inhibitor rather than directly inhibiting the protease.


Fig. 7. Fetal bovine serum inhibits the secretion of the Abeta -degrading metalloprotease by CHO cells. Confluent CHO cells were conditioned (37 °C, 24 h) in N2 medium in the absence (lane 2) or presence of FBS at concentrations of 1% (lane 3), 2% (lane 4), 4% (lane 5), 8% (lane 6), or 10% (lane 7). These CM were then incubated with the labeled 7PA2 medium for another 16 h, and the remaining amounts of Abeta analyzed as in Fig. 1. As controls, unconditioned N2 medium (lane 1) and medium from cells conditioned without serum to which 10% FBS was then added (lane 8) were also incubated with the labeled 7PA2 medium.
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Fig. 8. Stimulation of BV-2 cells by LPS enhances Abeta -degrading activity. A, RPMI and 10% FBS alone (lanes 1 and 2) or with CHO cells (lanes 3 and 4) or with BV-2 cells (lanes 5 and 6) were conditioned at 37 °C for 5 h in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of LPS. The CM were collected and Abeta -degrading activity detected as in Fig. 1. B, BV-2 cells were conditioned in the absence (lane 2) or presence (lanes 3 and 4) of LPS at 37 °C for 24 h. Unconditioned medium (lane 1) served as a negative control. 1,10-Phenanthroline was added to the aliquot in lane 4.
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To further characterize the regulation of the metalloprotease in the microglial line, E. coli LPS, a general stimulator of macrophage and microglial cells (14), was applied to the cells during conditioning in the presence of 10% FBS, since the latter generally decreased the amount of Abeta -degrading activity. LPS stimulated the BV-2 cells to release more Abeta -degrading protease, but failed to do so as expected in the CHO fibroblast line (Fig. 8A). As the conditioning time in LPS was prolonged, the stimulation of Abeta -degrading activity in BV-2 CM rose. The released protease was specifically blocked by 1,10-phenanthroline (Fig. 8B), suggesting that the protease activity stimulated by LPS in the presence of serum represented the same metalloprotease observed constitutively in the absence of serum.


DISCUSSION

The degree of Abeta deposition seen in AD and aged normal brains is determined by the rates of both Abeta production and Abeta removal. Therefore, it is important to understand the contribution of Abeta removal under normal and pathological circumstances, identify its mechanisms, and search for methods to enhance clearance. In this report, we describe a metalloprotease secreted by both non-neural and neural cells that is capable of efficiently cleaving Abeta and p3 but not oligomeric Abeta species found endogenously in the medium of beta APP-expressing cells.

Our studies using [35S]methionine-labeled CM of beta APP-transfected CHO cells as a source of Abeta and related species and the CM of different cell lines as a source of proteases show that several non-neural and neural cell lines secrete Abeta -degrading proteases, although the levels of activity vary substantially among cell types. Because the two culture media were incubated together in the absence of cells, our paradigm excludes the possibilities of cell-mediated internalization of Abeta (19, 20) or the degradation by a cell surface protease, if such exists. By using EDTA to lift the cells during passage, we also excluded the possibility that the serine protease/alpha 2-macroglobulin complex we previously described in vitro (15) was responsible for Abeta degradation. Two different methods, immunoprecipitation and ELISA, were employed to assess the amounts of Abeta remaining in the media after incubation and to confirm unequivocally that it was Abeta itself that was the substrate. We consistently observed the degradation of Abeta by proteases secreted by two non-neural cell lines, CHO and COS, and three neural cell lines, M17 and SY5Y neuroblastoma and BV-2 microglial cells (Fig. 1). Among them, BV-2 and CHO showed the strongest activity, followed in decreasing order by COS, M17, and SY5Y. BV-2, a murine microglial cell line, released the most Abeta -degrading activity per cell (Fig. 2), suggesting that microglial cells could play an important role in the clearance of Abeta from the extracellular space of brain. Furthermore, unlike the CHO fibroblast cells, which released little Abeta -degrading activity into medium in the first 6 h of conditioning, BV-2 cells immediately and continually secreted proteolytic activity after being placed in serum-free medium (Fig. 3).

The metalloprotease inhibitors 1,10-phenanthroline and EDTA substantially blocked the degradation of endogenous Abeta by the CM of both CHO and BV-2 cells (Fig. 3). Phosphoramidon, another metalloprotease inhibitor, which can only block certain metalloproteases (18), lacked the ability to inhibit the protease we describe. Selected inhibitors of the cysteine and aspartyl classes likewise produced little or no detectable inhibition of Abeta degradation under the conditions of our experiments. However, Pefabloc, a broad spectrum serine protease inhibitor, modestly inhibited the catabolism of Abeta by the conditioned media we examined (Fig. 3), suggesting that a serine protease may contribute to Abeta degradation in our cultures, although it plays a minor role. Naidu et al. (22) have reported that a mixture of inhibitors of all four major protease classes (leupeptin, pepstatin, EDTA, and phosphoramidon) inhibited Abeta degradation by CHO cells. In the cell lines we used, a metalloprotease is responsible for the major Abeta degradation, as confirmed when the specific Abeta -derived proteolytic products seen in acid-urea gels were completely abolished by 1,10-phenanthroline (Fig. 4). This inhibitor was not tested by Naidu et al.

Because the major class of secreted metalloproteases is the matrix metalloproteases, we have recently characterized both CHO and BV-2 CM by chromatography on gelatin-Sepharose, a matrix metalloprotease binding gel (23). The flow-through fractions of this column showed no loss of Abeta -degrading activity. Moreover, p-aminophenylmercuric acetate, a matrix metalloprotease activator, did not change the level of Abeta degradation of CHO and BV-2 CM, whereas it increased the degree of gelatin degradation by these CM, as shown by gel zymography.2 These results suggest that the protease we have characterized belongs to a class of metalloprotease different than the principal matrix metalloproteases.

Because accumulating studies have shown that Abeta 42 peptides are the initial species involved in the formation of amyloid deposits in AD and Down's syndrome (4, 8, 24, 25) despite the fact that cells secrete much more Abeta 40 than Abeta 42 (8), we characterized the specificity of the metalloprotease for secreted peptides ending at Abeta 40 and Abeta 42. Both CHO and BV-2 CM decreased the amounts of total Abeta and Abeta 42 to similar levels and left the ratio of Abeta 42 to total Abeta largely unchanged (Fig. 5), suggesting that the metalloprotease has equal avidity for endogenous Abeta 1-40 and Abeta 1-42 peptides. We found that the p3 peptide (Abeta 17-40/42) was more resistant to degradation by the metalloprotease than Abeta (Fig. 6). Interestingly, the low molecular weight Abeta oligomers that can be detected in CHO medium (13) and that are composed of both Abeta 40 and Abeta 42 peptides3 were unchanged after 48 h incubation in conditions that allowed virtually complete degradation of monomeric Abeta and p3. Therefore, we speculate that the threshold concentration of monomeric Abeta needed to allow aggregation could be reached by increased production and/or impaired degradation of Abeta . After Abeta 40 and Abeta 42 peptides are oligomerized, they will apparently be cleared inefficiently by secreted proteases, even those released by activated microglial cells in senile plaques. These oligomers could then accumulate and ultimately form high molecular weight assemblies, including the potentially neurotoxic amyloid fibrils characteristic of AD.

Because the clearance of Abeta should help determine the levels of extracellular Abeta and thus the rate of fibril formation, methods to stimulate Abeta degradation represent one approach to slowing the development of AD neuropathology. During our experiments, we noticed that the activity of Abeta -degrading metalloprotease decreased in parallel with increasing levels of serum in the cultures (Fig. 7), suggesting that the enzyme is probably regulated by extracellular factors under physiological conditions. It is currently unclear whether this loss of activity is caused by the increased secretion of a protease inhibitor by the cells or by decreased release of the protease itself. We also found that LPS, a general stimulator of macrophage/microglia, activated BV-2 cells to increase Abeta -degrading metalloprotease activity in their media under the proteolytically adverse condition of culturing in 10% serum (Fig. 8). It is possible that the activated microglia which are consistently found in mature (neuritic) plaques but less frequently present in immature (diffuse) plaques (21) release proteases such as that described here. Our data lead to the hypothesis that at early stages of AD, microglial cells could play an active role in slowing the development of Abeta deposits and amyloidosis; however, at later stages there could be an adverse effect, because the released metalloprotease cannot efficiently degrade oligomeric Abeta but would be available to act upon other cellular and extracellular substrates, thereby potentially aggravating the inflammatory and cytotoxic events in and around the mature plaque. Full purification of the metalloprotease should allow the development of antibodies and probes useful for examining the role of this and related proteases in the normal and abnormal biology of Abeta .


FOOTNOTES

*   This work was supported by National Institutes of Health Grants AG 06173 and AG 12749 and grants from the Richard Saltonstall Charitable Trust and the Alder Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: Brigham and Women's Hospital 221 Longwood Ave., LMRC 103, Boston, MA 02115. Tel.: 617-732-6454; Fax: 617-732-7787; E-mail: selkoe{at}cnd.bwh.harvard.edu.
1   The abbreviations used are: AD, Alzheimer's disease; Abeta , amyloid beta  protein; beta APP, beta -amyloid precursor protein; CHO, Chinese hamster ovary; FBS, fetal bovine serum; CM, conditioned medium; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharide; Tricine, N-tris(hydroxymethyl)methylglycine.
2   W. Q. Qiu and D. J. Selkoe, unpublished data.
3   M. B. Podlisny and D. J. Selkoe, unpublished data.

Acknowledgments

We are indebted to Dr. V. Bocchini for providing the BV-2 cells. We thank Drs. D. Walsh, D. Teplow, and H. A. Chapman for suggestions and helpful discussions.


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