(Received for publication, June 30, 1994; and in revised form, November 10, 1994)
From the
The primary histopathological feature of Alzheimer's
disease is the accumulation of -amyloid in the brains of afflicted
individuals. This peptide has been shown to be produced and liberated
both in vitro and in vivo by normal physiological
processes. The mechanism by which
-amyloid is formed, as well as
that by which it may be cleared, are events likely to impact on the
development and progression of this disease. Thus, the fate of
-amyloid peptides secreted by cultured mammalian cells was
investigated. It was found that levels of the soluble peptide are
reduced over time due to the activity of multiple types of proteinases
including those from the metallo, aspartyl, and thiol classes.
Inhibitors to each class of proteinase can only partially block
-amyloid degradation, but, if used in combination, they can fully
prevent its catabolism. The Kunitz serine proteinase inhibitor domain,
present on two
-amyloid precursor protein isoforms, was found to
be an effective inhibitor of
-amyloid peptide degradation. These
data indicate that modulations in expression of secreted proteinases
and/or
-amyloid precursor isoforms may influence levels of
-amyloid.
Deposits of -amyloid peptide are characteristic of aging
and Alzheimer's disease. The
-amyloid peptide, at
sufficiently high concentrations, has a propensity to aggregate into
insoluble precipitates. This 39-43 amino acid peptide is derived
from a larger precursor, the
-amyloid precursor protein
(
-APP) (
)which can be expressed in multiple isoforms.
The two major isoforms are distinguished by the presence (751 amino
acids) or absence (695 amino acids) of a serine proteinase inhibitor
domain of the Kunitz family
(KPI)(1, 2, 3, 4) .
Alzheimer's disease-specific increases in the expression of KPI
domain-bearing
-APP isoforms have been observed in hippocampal and
cortical neurons(5) , as well as in whole
brain(6, 7, 8) . These data suggest that an
imbalance in
-APP isoform expression and/or an excess of KPI may
be pathogenic.
Recently, it has been shown that -amyloid
peptide is a naturally generated soluble protein. The peptide has been
detected in the medium of a variety of cultured mammalian
cells(9, 10, 11) , as well as in serum and
cerebrospinal fluid(12) . The
-amyloid peptide seems to be
a minor degradation product of
-APP catabolism, the production of
which appears directly correlated with the expression of excess or
aberrant
-APPs(13) .
While the biology of -amyloid
formation in vitro has been a focus of study, the fate of this
peptide after it is secreted from the cell has not been examined. Both
the synthesis and clearance of this protein may potentially contribute
to increased levels of
-amyloid and to the development of
insoluble deposits. Therefore, to begin to understand the catabolic
aspects of
-amyloid biology we characterized the turnover of this
protein produced in vitro.
A clonal line of cultured Chinese hamster ovary fibroblasts
which was stably transfected and expresses the human 695-amino acid
isoform of -APP (termed CP-6) was used as a source of
-amyloid protein. Based on recent reports, cultured mammalian
cells have been demonstrated to produce and secrete
-amyloid
peptides which include an intact 4-kDa
-amyloid peptide and an
amino-terminally truncated 3-kDa peptide derived from the secretory
cleavage of
-APP(5, 6, 7, 15) .
The single methionine residue present in the carboxyl-terminal domain
of the
-amyloid peptide permits metabolic labeling with
[
S]methionine of both the 3- and 4-kDa
-amyloid peptides.
To examine the temporal profile of
-amyloid expression, we employed a pulse-chase experimental
format. Multiple cultures of CP-6 cells were metabolically radiolabeled
for 1 h by addition of [
S]methionine/cysteine to
the medium. Following the pulse period, the isotope-containing medium
was removed, and the cell cultures were replenished with fresh medium
lacking isotope and incubated. At 2, 4, 6, 8, 10, and 12 h post-pulse,
the conditioned medium was harvested. The fate of
[
S]methionine-labeled
-amyloid peptides
secreted into the culture medium over the 12-h incubation period was
followed by immunoprecipitation with a
-amyloid-specific antiserum
and electrophoresis on a 16.5% Tris/Tricine/polyacrylamide gel. Fig. 1displays the results from the pulse-chase experiment.
Maximum accumulation of both the 4- and 3-kDa
-amyloid peptides
was observed 6 h into the chase which remained high for 2 h then
declined. By 12 h after pulse-labeling, modest amounts of the
-amyloid peptides remained. When the chase period was extended to
include a 24-h time point, little
-amyloid was detected (data not
shown). It should be noted that CP-6 cells were viable and
metabolically active during the 24-h serum-free treatment, and,
therefore, this was not a factor in the observed reduction of
-amyloid peptides. In fact, levels of several labeled
cell-associated proteins remained unchanged over the 24-h chase period
indicating general cellular proteolysis mediated by serum deprivation
was not responsible for the observed decrease in
-amyloid
peptides. The time to eliminate the bulk of
[
S]methionine-labeled
-amyloid peptides
from the serum-free medium after maximum levels are reached was
determined using densitometry and found to be >12 h. Similar results
were obtained for identical experiments performed with CP-6 cells of
different passage.
Figure 1:
-Amyloid peptide turnover in
cell-conditioned medium. Multiple dishes of CP-6 cells were
pulse-labeled with [
S]methionine/cysteine for 1
h after which label was removed and isotope-free medium was added. At
2, 4, 6, 8, 10, and 12 h into the chase period, medium was harvested
and analyzed for [
S]methionine-labeled
-amyloid using immunoprecipitation, polyacrylamide gel
electrophoresis (PAGE), and
autoradiography.
The secretion and elimination of the
-amyloid peptides by CP-6 cells was analyzed using a different
assay, a radioimmunoassay. This radioimmunoassay employs a second
-amyloid-specific antiserum from that used for
immunoprecipitation. Each serum recognizes a different epitope within
the carboxyl-terminal region of the
-amyloid sequence.
Consequently, both the 4- and 3-kDa peptides are scored in both assays.
The secretion of soluble
-amyloid peptides into serum-free culture
medium was followed utilizing the radioimmunoassay. Identical with the
results obtained by immunoprecipitation, the radioimmunoassay revealed
peak levels of peptide
6 h into the time course followed by a
substantial decrease in levels by 24 h (Fig. 2). Some variation
from experiment to experiment was noticed, however, in the
-amyloid peptide level at 24 h. Generally, 10-30% of maximum
levels were present at 24 h; however, in one experiment, as much as 50%
remained. Approximately 2.5 ng/ml
-amyloid protein was present in
the conditioned medium at peak times (
6 h) which is roughly
equivalent to the level described that was released from a human brain
primary cell culture(12) . Similar to a previous
report(7) , the absolute level of
-amyloid protein,
however, fluctuated with cell type. Levels also varied slightly with
cell passage.
Figure 2:
Quantitation of -amyloid peptide
turnover in cell-conditioned medium.
-Amyloid in CP-6 cultured
cell media which were prepared in Fig. 1was quantitated by film
densitometry and phosphorimaging (open circles). An identical
time course experiment was performed as in Fig. 1except that
radioisotope was omitted and media samples were scored for
-amyloid using a radioimmunoassay (filled
diamonds).
These experiments, assayed by either
immunoprecipitation or radioimmunoassay, indicated that the secreted
-amyloid peptides were depleted from the culture medium over time.
To determine whether the peptides were removed due to cellular uptake
or to proteolytic degradation, we investigated the potential catabolic
effects of conditioned medium on
-amyloid peptides in the absence
of cells by performing mixing experiments. Medium which had been
conditioned by CP-6 cells for 24 h was mixed with either unlabeled or
[
S]methionine/cysteine-labeled serum-free medium
harvested 4 h after addition to the cell monolayer. 4-h conditioned
medium represents a time when levels of soluble
-amyloid peptide
were high. The combined 24- and 4-h media (1:1 volume) were incubated
at 37 °C, in the presence or absence of proteinase inhibitors,
after which the mixed media were assayed for
-amyloid protein. As
a control treatment, 4-h conditioned medium was mixed (1:1 volume) with
medium never exposed to cells. By performing a time course for the 37
°C treatment, it was determined that
25 h was a desirable
incubation period (data not shown) and was, consequently, used for all
experiments.
The data presented in Fig. 3are a compilation
of multiple mixing experiments using unlabeled media followed by
radioimmunoassay scoring of -amyloid protein. When 4-h CP-6
cell-conditioned medium was combined with control medium and incubated
at 37 °C for 0 or for 25 h, the level of
-amyloid protein was
unchanged and remained at
3 ng/ml. In contrast, when the 4-h
medium was incubated with 24-h conditioned medium, the amount of
-amyloid protein was reduced by greater than 50% after the 25-h
incubation. No depletion was observed with 4- and 24-h media which were
mixed but not incubated. An increase of
-amyloid protein (
1
ng/ml) was frequently measured for the unincubated 4- plus 24-h sample
over the level present in only 4-h medium. It is probable that this
increase was derived from residual
-amyloid peptides present in
the 24-h medium which, when mixed with the 4-h medium, resulted in
slightly higher initial amounts. Significantly, a reduction in
-amyloid protein after incubation with 24-h conditioned medium was
prevented by the addition of a collection of proteinase inhibitors. The
mixture of inhibitors (mix-C) used and the concentration of each is
summarized in Table 1. Since protection of
-amyloid peptides
was seen in the presence of proteinase inhibitors, these data suggest
that proteolytic enzymes were involved in the observed reduction of
this protein. It should be noted that on occasion some samples of 4-h
CP-6 conditioned media showed slight
-amyloid peptide catabolic
activity; however, most preparations lacked this effect. This suggests
that the degrading proteolytic enzymes were not released or
sufficiently accumulated in the medium until after 4 h.
Figure 3:
Degradation of -amyloid peptides by
cell-conditioned medium and protection by proteinase inhibitors. Medium
conditioned by CP-6 cells for 4 h was incubated at 37 °C with
control medium (EMEM) or medium conditioned by CP-6 cells for
24 h, either in the presence or absence of proteinase inhibitor mix-C (PI mix) (refer to Table 1). Incubations were for 0 h (thin-lined columns) or for 25 h (large line cross-hatched
columns) after which
-amyloid was measured by
radioimmunoassay.
The above
result was confirmed and extended in experiments using 4-h conditioned
media containing [S]methionine-labeled
-amyloid peptides and an immunoprecipitation assay (Fig. 4). Again, the
-amyloid peptides were depleted after
incubation at 37 °C for 25 h with 24-h CP-6 cell-conditioned medium
but, if incubated in the presence of the proteinase inhibitor mixture,
protection against degradation was observed. Both the 4- and 3-kDa
peptides were degraded in the absence of proteinase inhibitors. No
significant reduction in the
[
S]methionine-labeled peptides was seen when the
sample was identically incubated with control medium.
Figure 4:
Degradation of
[S]methionine-labeled
-amyloid peptides by
cell-conditioned medium and protection by proteinase inhibitors.
[
S]Methionine-labeled
-amyloid peptides
present in 4-h CP-6 cell-conditioned medium (CM) was incubated
at 37 °C for 25 h with control medium (control CM) or with
24-h unlabeled CP-6 cell-conditioned medium (24hr CM), either
in the presence or absence of proteinase inhibitor mix-C (+PI
mix-C) (refer to Table 1). After incubation,
-amyloid
protein remaining in media samples was analyzed by immunoprecipitation,
PAGE, and autoradiography.
The
proteolytic activity released by CP-6 fibroblasts which degrades the
-amyloid peptides was found to be present in media conditioned by
other cell types. Fig. 5illustrates this point in which 24-h
conditioned medium was prepared from a human neuroblastoma cell line,
SK-N-MC, and tested. The SK-N-MC conditioned medium was mixed with 4-h
CP-6 medium, incubated at 37 °C for 25 h in either the presence or
absence of proteinase inhibitors described in Table 1(mix-C).
The SK-N-MC medium promoted a 50% reduction in
-amyloid peptides
which was prevented by the addition of the collection of proteinase
inhibitors.
Figure 5:
Degradation of -amyloid peptides by
SK-N-MC cell-conditioned medium. Medium conditioned for 4 h by CP-6
cells was incubated at 37 °C for 0 h (darker columns) or
for 25 h (light cross-hatched columns) with medium conditioned
for 24 h by SN-N-MC neuroblastoma cells either in the presence (PI
Mix) or absence of proteinase inhibitors described in Table 1under mix-C. The
-amyloid present after incubation
was measured by radioimmunoassay.
To identify the type of proteinase(s) responsible for
the clearance of the -amyloid peptides, we examined the inhibitory
effects of individual proteinase inhibitors present in the mixture.
This inhibitor mixture consisted of a number of commonly used
proteinase inhibitors covering the four major classes of proteinases;
serine, metallo, aspartyl, and thiol. A standard concentration of each
inhibitor was used for the mixture. When each inhibitor was
individually evaluated for its ability to block
-amyloid peptide
degradation using analysis by radioimmunoassay, none was effective
(data not shown). This result suggested that multiple proteinases may
be involved in the degradation of these proteins. Therefore, we studied
the effects of inhibitor combinations. These combinations were based on
class or classes of enzyme target. The inhibitor combinations tested
are outlined in Table 1. We repeatedly found that a mixture of
leupeptin and pepstatin partially protected the breakdown of
-amyloid and that the addition of EDTA and phosphoramidon to this
mixture fully protected the
-amyloid peptides ( Fig. 6and Table 2). Other proteinase inhibitor combinations proved
ineffective ( Fig. 6and data not shown). To provide further
evidence that enzymes are responsible for the protein depletion, we
characterized the effect of boiled 24 h conditioned CP-6 medium on the
catabolism of the
-amyloid peptides. No degradation was seen when
4-h medium was incubated at 37 °C for 25 h with boiled 24-h medium (Table 2).
Figure 6:
Protection of -amyloid peptide
degradation by select proteinase inhibitors. CP-6 cell medium
conditioned for 4 h (4hr) was incubated at 37 °C for 0 h
or for 25 h with control medium (EMEM) or with CP-6 cell
medium conditioned for 24 h (24hr) to which combinations of
proteinase inhibitors (PI mix) were added. Refer to Table 2for composition of individual inhibitor mixes.
-Amyloid protein remaining in each sample was measured by
radioimmunoassay. Values are expressed as the percentage of
-amyloid protein present in each sample after 25 h of incubation
compared to no incubation or ``0 hr''
incubation.
The inhibitory effects seen with leupeptin,
pepstatin, EDTA, and phosphoramidon indicated that thiol, aspartyl, and
metalloproteinases together comprise the -amyloid peptide
degrading activity. Since leupeptin can also inhibit select serine
proteinases, in addition to thiol proteinases, we were interested in
determining whether the KPI domain, a serine proteinase inhibitor,
harbored within the 751- and 770-amino acid isoforms of
-APP could
block the breakdown of
-amyloid peptides. We have previously
described the recombinant production, purification, and activity of the
57-amino acid KPI domain derived from
-APP(14, 16) . A mixing experiment was performed
in which [
S]methionine-labeled
-amyloid
peptides present in 4-h conditioned CP-6 medium were incubated with
24-h conditioned medium in the absence or presence of 50 µM KPI. After the standard incubation, the radiolabeled
-amyloid
peptides were immunoprecipitated and analyzed by polyacrylamide gel
electrophoresis (Fig. 7). Partial protection (quantified at 50%)
was found upon addition of the
-APP Kunitz inhibitor domain to the
mixed media. In the absence of KPI inhibitor, the 3- and 4-kDa
-amyloid peptides were completely eliminated.
Figure 7:
Partial protection of -amyloid
peptide degradation by
-APP KPI domain.
[
S]Methionine-labeled
-amyloid peptides
present in 4-h medium conditioned and labeled by CP-6 cells was mixed
with control medium (control CM) or with unlabeled medium
conditioned by CP-6 cells for 24 h (24hr CM). Replica samples
of the 4-h plus 24-h mixture were incubated in the presence (24hr
CM + KPI) or absence (24hr CM) of 50 µM KPI domain. Following incubation of all samples at 37 °C for
25 h,
-amyloid protein was analyzed by immunoprecipitation, PAGE,
and autoradiography.
Accumulation of -amyloid peptide appears to have a
central role in the pathogenesis of Alzheimer's disease.
Substantial evidence, both direct and indirect, implicate this protein
as a major contributing factor in the initiation and progression of
this disorder (reviewed in (17) and (18) ). Several
properties of the
-amyloid peptide are of particular relevance to
Alzheimer's disease, notably the ability to self-polymerize
(reviewed in (19) and (20) ) and to induce
neurotoxicity (21; reviewed in (20) and (22) ). These
two properties are also inter-related in that
-amyloid
neurotoxicity is directly correlated with the aggregation state of the
peptide(23, 24) . It has been shown that
-amyloid
peptide is generated as part of normal cellular physiology (9, 10, 11, 12) and presumably as a
result of
-APP catabolism(13) . As part of this cellular
process, the
-amyloid peptide is secreted from the cell as a
soluble protein. In addition to the extracellular soluble nature of
-amyloid peptide, the local concentration of this protein in the
microenvironment is likely to be an important factor in eliciting
neuronal degeneration and plaque formation.
Because increases in
soluble -amyloid peptide are likely to enhance its propensity for
self-polymerization, it is important to understand the fate of
-amyloid peptide after it is released from the cell. Heretofore,
studies have primarily focused on the processes of
-amyloid
peptide formation and not on its clearance. Therefore, to begin to
understand the overall biology of
-amyloid peptide, we
investigated the turnover profile of this protein produced in
vitro.
Our studies, using cultured mammalian cells as a source
of -amyloid peptide, indicate that this protein is degraded within
12 to 24 h of its release from the cell. Two different methods and
immunoreagents were employed to measure
-amyloid peptide, a
radioimmunoassay and an immunoprecipitation assay. The data obtained
from both assays were in complete agreement. Catabolism of both the
4-kDa
-amyloid peptide and the 3-kDa truncated form of this
protein was found to be due to proteolytic activity released by the
cultured cells. Chinese hamster ovary fibroblastic and human SK-N-MC
neuroblastoma cell lines were characterized as having proteolytic
activities which degrade the
-amyloid peptides. Serum-free medium
which had been conditioned by the cultured mammalian cells was the
source of the
-amyloid degrading activity employed in these
studies. Therefore, the elimination of
-amyloid was not due to
reinternalization by the cell. Cellular uptake of exogenously supplied
-amyloid peptide has been described(25) .
The
-amyloid catabolic activity was shown to be of enzymatic nature in
that it could be fully inhibited by heat denaturation or by the
addition of proteinase inhibitors. Several classes of proteinases were
identified as being responsible for the breakdown of the
-amyloid
peptide based on the types of inhibitors found to be effective in
preventing degradation. Of the active families of proteinases secreted
by the cells studied, thiol, aspartyl, and metalloproteinase members
appeared responsible for
-amyloid catabolism. Select inhibitors of
each class were only partially able to prevent
-amyloid
degradation. Only when these multiple types of proteinase inhibitors
are used collectively could
-amyloid catabolism be fully blocked
within the selected incubation period. In addition to the above
mentioned proteinases, it appeared that a serine proteinase(s) also
contributed to
-amyloid turnover since the KPI domain of
-APP
could partially block this process. However, this KPI-responsive
activity appears unique in that other serine proteinase inhibitors such
as aprotinin, TLCK, and benzamidine were incapable of blocking
-amyloid peptide degradation. Likewise, one thiol proteinase
inhibitor was ineffective in protecting
-amyloid clearance,
whereas another inhibitor of this class was effective.
As mentioned,
the serine proteinase inhibitor domain, KPI, contained in some
-APP isoforms was tested and was found to partially protect
(
50%) the degradation of
-amyloid. That the KPI domain of
-APP can partially inhibit the degradation of
-amyloid
peptide in vitro may hold relevance to the increase in KPI
containing
-APP isoforms specifically observed in
Alzheimer's disease brain
tissue(5, 6, 7, 8) . Furthermore, in
one study, the increase in
-APP isoforms containing the KPI domain
was directly correlated with the density of plaques(5) . Since
-APP has been shown to be secreted from the cell both in vitro(26) and in vivo(27) , such an increase
in expression of KPI bearing
-APP isoforms in the disease state
might lead to increased levels of secreted KPI
-APPs. Higher
levels of soluble KPI-containing
-APP might retard the clearance
of
-amyloid peptide and, in turn, contribute to increased local
concentrations of
-amyloid and to the development of insoluble
aggregates. It may be relevant that secreted
-APP has been
reported to harbor a metalloproteinase inhibitor domain (28) which might also serve to retard
-amyloid catabolism
and promote deposit formation.
Many factors might influence the
normal clearance rate of -amyloid protein in addition to the
possible contribution of increased expression of
-APP and its
resident serine and metalloproteinase inhibitory activities. The
binding of
-amyloid to lipoproteins apoE (29) demonstrated with in vitro studies might
sequester this protein, thereby arresting or retarding its degradation.
In addition, it is possible that aberrant expression or bioactivity of
any of the
-amyloid degrading proteinases might have a
pathological contribution by elevating levels of this protein. Hence,
knowledge of the
-amyloid degrading proteinases may be important
to our complete understanding of
-amyloidogenesis and
Alzheimer's disease.