(Received for publication, December 4, 1996, and in revised form, December 27, 1996)
From the Center for Neurologic Diseases, Harvard Medical School,
Brigham and Women's Hospital, Boston, Massachusetts 02115 and
Athena Neurosciences, Inc.,
South San Francisco, California 94080
Amyloid -protein (A
) 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
-amyloid precursor protein and on the
production and polymerization of A
, understanding the degradation
and clearance of A
has received very little attention. By incubating
the conditioned medium of metabolically labeled A
-secreting cells
with media of various cultured cell lines, we observed a
time-dependent decrease in the amount of A
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 A
-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
A
1-40 and A
1-42 peptides secreted by
-amyloid precursor protein-transfected cells but failed to degrade
low molecular weight oligomers of A
that form in the culture medium.
Lipopolysaccharide, a stimulator of macrophages/microglia, activated
BV-2 cells to increase their A
-degrading metalloprotease activity.
We conclude that secreted A
1-40 and
A
1-42 peptides are constitutively degraded by a
metalloprotease released by microglia and other neural cells, providing
a potential mechanism for the clearance of A
in brain tissue.
The defining pathological features of Alzheimer's disease
(AD)1 are extracellular deposits of amyloid
-proteins (A
) that form senile plaques and amyloid angiopathy and
intraneuronal deposits of modified tau proteins that form
neurofibrillary tangles. A
are 40-43-amino acid proteolytic
fragments generated by unidentified proteases from the transmembrane
glycoprotein,
-amyloid precursor protein (
APP) (1, 2). The gene
encoding
APP is on human chromosome 21q, and missense mutations in
and around the A
coding region of this gene are a rare cause of
familial AD (FAD). Moreover, trisomy 21 (Down's syndrome) is
characterized by overexpression of
APP due to increased gene dosage,
resulting in very early A
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 A
(8, 9). These and other findings provide strong evidence that disordered
APP metabolism can increase the production of A
peptides, particularly the A
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
APP progressively develop A
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 APP by cells in order to understand how A
is generated normally and in AD. In contrast, little is known about how
A
, once secreted, is degraded and cleared from tissues. At present,
only the relatively rare forms of FAD linked to the
APP or
presenilin genes are thought to involve overproduction of A
. The
excessive cerebral accumulation of A
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 A
. If specific A
-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 A peptides naturally secreted by cells. We have
identified a secreted metalloprotease activity that efficiently degrades endogenous A
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 A
40 and
A
42 monomers than A
oligomers formed in cell culture.
These findings have implications for the normal and pathological
clearance of A
in brain.
Chinese hamster ovary (CHO) cells stably
transfected with APP770 cDNA containing the Val
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.
To obtain conditioned
media (CM) containing A-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 A
-degrading activity,
confluent monolayers of 7PA2
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 A
remaining in each sample was assessed by
immunoprecipitation with the high affinity A
antibody, R1282 (4).
This antibody was generated to synthetic A
1-40 peptide
and characterized similarly to our A
antibody, R1280, used in our
previous study (15).
A degradation in cultures was also quantified by incubating 7 µl
of conditioned or unconditioned medium with 125I-labeled
A
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.
|
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 A or specific A
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 A
), for total A
or 21F12 (raised against
residues 33-42 of A
), for A
peptides ending specifically at
residue 42. The reporter antibody was 3D6 (to residues 1-5 of A
) in
both assays. The ratio of A
1-42 over total A
in each
sample was calculated.
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 A-degrading activity as described above.
We examined several non-neural and neural cell
lines for the secretion of an A-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/
2-macroglobulin complex in the medium, which we
recently showed can occur during tryptic passage of cells and degrade
secreted A
(15). To search for A
-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 A
, CHO cells stably
transfected with
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 A
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 A
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 A
antibody
(R1282) and performing gel fluorography, we observed variable declines
in the amounts of A
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 A
after incubation (Fig. 1).
To obtain quantitative information about A 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 A
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 A
-degrading activity. Conditioning these cell lines for increasing intervals revealed that the confluent CHO cells produced
little or no A
-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.
A
To determine whether that the loss
of A 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
A
1-40 peptide (40 ng/ml) as a substrate, followed by a
sandwich ELISA to detect remaining intact A
. The inhibitor profile
clearly demonstrated an A
-degrading metalloprotease in the CHO
medium (Table I). The degradation of cell-secreted A
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) A
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 A
in this system (Fig. 3). These results
indicate that the major A
-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 A
degradation (Fig. 3), suggesting that a serine
protease may also make a small contribution to A
proteolysis by CHO
and BV-2 media.
To detect the products of the A degradation, acid-urea-PAGE (15) was
used to analyze 125I-labeled synthetic
A
1-40 after incubation in CHO or BV-2 CM for 24 h
(Fig. 4). The amount of intact A
decreased markedly, and 3 peptide bands appeared that migrated differently from intact A
and were not produced by incubation in N2 medium alone. The production
of these fragments was completely inhibited by 1,10-phenanthroline, confirming that A
is degraded by a secreted metalloprotease in both
CHO and BV-2 CM and generates similar peptide products.
Degradation of A
Because A peptides ending at
residue 42 (A
1-42) are the initially deposited species
in AD and normal aged brain tissue, we examined the substrate
specificity of the metalloprotease on endogenous A
1-40
versus A
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 A
(including A
1-40 and A
1-42)
or A
1-42 alone. The amounts of total A
and
A
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
A
1-42 to total A
showed only a slight and
insignificant increase (Fig. 5C).
We also characterized the specificity of the metalloprotease in the
24-h conditioned media of CHO and BV-2 cells for A-related species
found in the culture medium of our
APP-expressing 7PA2 cells. A
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
A
species that we previously documented in the CM of these V717F
APP-transfected CHO cells (13) were completely resistant to
proteolysis (Fig. 6).
Regulation of the Secreted Metalloprotease
During the course
of these studies, we noticed that high A-degrading activity in CM
(>90% decrease of A
compared to that in N2) occurred when cells
were conditioned in the absence of serum. In contrast, only a 10-15%
decrease in A
occurred when 10% FBS was present during conditioning
(data not shown). To clarify the role of serum in the production and
activity of the A
-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 A
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 A
as much as CHO CM without FBS, whereas
the amount of A
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 A
-degrading activity (Fig. 7). Using the ELISA assay,
we were similarly able to detect only a 10-20% decrease in A
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 A
proteolysis.
On the other hand, adding 10% FBS solely during the incubation period
did not decrease the strong A
-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 A
-degrading
metalloprotease or increase the secretion of a metalloprotease inhibitor rather than directly inhibiting the protease.
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 A-degrading activity. LPS stimulated the
BV-2 cells to release more A
-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 A
-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.
The degree of A deposition seen in AD and aged normal brains is
determined by the rates of both A
production and A
removal. Therefore, it is important to understand the contribution of A
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 A
and p3 but
not oligomeric A
species found endogenously in the medium of
APP-expressing cells.
Our studies using [35S]methionine-labeled CM of
APP-transfected CHO cells as a source of A
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 A
-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 A
(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/
2-macroglobulin complex we previously described in vitro (15) was responsible for A
degradation. Two
different methods, immunoprecipitation and ELISA, were employed to
assess the amounts of A
remaining in the media after incubation and to confirm unequivocally that it was A
itself that was the
substrate. We consistently observed the degradation of A
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 A
-degrading activity per
cell (Fig. 2), suggesting that microglial cells could play an important
role in the clearance of A
from the extracellular space of brain. Furthermore, unlike the CHO fibroblast cells, which released little A
-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 A 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 A
degradation under the conditions of our experiments. However, Pefabloc,
a broad spectrum serine protease inhibitor, modestly inhibited the catabolism of A
by the conditioned media we examined (Fig. 3), suggesting that a serine protease may contribute to A
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 A
degradation by CHO cells. In the cell lines we used, a
metalloprotease is responsible for the major A
degradation, as
confirmed when the specific A
-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 A-degrading activity. Moreover,
p-aminophenylmercuric acetate, a matrix metalloprotease
activator, did not change the level of A
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 A42
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 A
40 than A
42
(8), we characterized the specificity of the metalloprotease for
secreted peptides ending at A
40 and A
42.
Both CHO and BV-2 CM decreased the amounts of total A
and
A
42 to similar levels and left the ratio of
A
42 to total A
largely unchanged (Fig. 5), suggesting
that the metalloprotease has equal avidity for endogenous
A
1-40 and A
1-42 peptides. We found that
the p3 peptide (A
17-40/42) was more resistant to
degradation by the metalloprotease than A
(Fig. 6). Interestingly,
the low molecular weight A
oligomers that can be detected in CHO
medium (13) and that are composed of both A
40 and
A
42 peptides3 were unchanged
after 48 h incubation in conditions that allowed virtually
complete degradation of monomeric A
and p3. Therefore, we speculate
that the threshold concentration of monomeric A
needed to allow
aggregation could be reached by increased production and/or impaired
degradation of A
. After A
40 and A
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 A should help determine the levels of
extracellular A
and thus the rate of fibril formation, methods to
stimulate A
degradation represent one approach to slowing the
development of AD neuropathology. During our experiments, we noticed
that the activity of A
-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 A
-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 A
deposits
and amyloidosis; however, at later stages there could be an adverse
effect, because the released metalloprotease cannot efficiently degrade
oligomeric A
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 A
.
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.