(Received for publication, November 4, 1994; and in revised form, December 15, 1994)
From the
Treatment of human 293 cells transfected with amyloid precursor
protein (APP) (the ``Swedish''
mutation) with a specific inhibitor of the vacuolar
H
-ATPases, bafilomycin A1 (baf A), leads to a potent
inhibition of the release of the A
peptide. This is accompanied by
a selective inhibition of
-secretase activity. Surprisingly, baf A
did not inhibit the production of A
from either wild-type APP (WT
APP) or from APP
(the ``Hardy'' mutation),
expressed in the same cell type. In contrast, the robust production of
A
from a human neuroglioma-derived cell line (HS683) transfected
with WT APP, or from primary human mixed brain cultures (HMBC)
expressing genomic WT APP, were also effectively inhibited by baf A.
The inhibition of A
production from the HMBC was also accompanied
by the inhibition of
-s-APP release. No inhibition of
-s-APP
release was seen in any of the cell types tested. These results
indicate that intracellular acidic processes are rate-limiting for
-secretase cleavage and A
production from SW APP, but not WT
APP, in the peripheral 293 cell line. Furthermore, such acidic
processes also play a rate-limiting role in A
release from human
central nervous system-derived cells, including HMBC. Differential
trafficking of the SW APP into an acidic compartment conducive to
-secretase cleavage and A
release could be one explanation
for the increased production of A
observed on expression of this
mutation.
The pathogenesis of Alzheimer's disease involves the
deposition of the A peptide as an early, and perhaps causative,
event in the subsequent maturation of such deposits into senile or
neuritic plaques (1) . The secretion of A
by human mixed
brain cultures (HMBC)(
)(2) , as well as a number of
APP-transfected cell lines in culture(3, 4) , suggests
that A
is produced by a normal metabolic pathway of the cell.
However, the cellular mechanisms that lead to the production of A
from APP are not well understood. One metabolic fate of newly
synthesized APP appears to be constitutive secretion of the bulk of its
ectodomain as
-s-APP(5) , after an endoproteolytic
non-amyloidogenic cleavage by an unidentified enzyme,
``
-secretase''(6) . However, other cellular
processing pathways must also contribute to APP metabolism, since a
substantial proportion of APP metabolism results in lysosomal
degradation of the mature, full-length
protein(7, 8, 9) . Although intermediates of
APP degradation containing the entire A
peptide region accumulate
in the cell upon treatment with lysosomal cysteine protease
inhibitors(8) , such inhibitors do not have any effect on
A
production(10) . Considerable doubt is thus cast upon
the relevance of such ``potentially amyloidogenic''
intermediates to the formation of A
. More relevant to A
formation, however, are observations on the analysis of a truncated
secreted form of APP that appears to terminate at Met-596
(
-s-APP)(11) . These forms, produced at a relatively low
level by the 293 cell line transfected with WT APP, form a larger
proportion of the total s-APP produced by HMBC, which correlates well
with the higher level of A
production in the primary cultures.
This suggests that an alternate enzymatic activity, dubbed
``
-secretase,'' is responsible for the production of the
truncated secreted form, and, by inference, a cell-associated
carboxyl-terminal fragment starting at Asp-597. Subsequent cleavage at
Val-637 or Ala-639 in the transmembrane domain of such a fragment by a
second protease would then release A
into the extracellular
medium.
We have utilized 293 cells stably transfected with either WT
APP751 or APP-NL, incorporating the so-called ``Swedish''
mutation (APP), as a system for investigating the
role of such an alternative secretory cleavage in the cellular
production of A
peptide. Tissue culture expression of this double
mutation, identified as pathogenetic for Alzheimer's disease in
the affected kindred(12) , results in a substantial stimulation
of A
production (13) , without any significant effect on
total s-APP released into the CM. Direct sequencing of the A
produced by these cells shows that the primary product is A
1-40(14) , indicating that the presumptive site of
-secretase cleavage has not been altered by the presence of the
double mutation. Preliminary data indeed suggested that in comparison
to wild-type APP751, transient transfections incorporating the Swedish
mutation produce more of a truncated s-APP (data not shown). This form
was not immunoprecipitable with antibodies that recognize epitopes
specific to the amino-terminal region of the A
peptide, consistent
with the previously described truncated cleavage. The relatively high
levels of A
produced by stable transfectants incorporating the
Swedish mutation allowed the design of experiments that tested the
ability of specific compounds to alter or affect the relative balance
between normal secretase cleavage and
-secretase cleavage. We were
especially interested in identifying whether
-secretase and
-secretase activity are separate manifestations of the same
enzymatic process, or whether they occur in different intracellular
compartments. Although acidotropic amines have previously been shown to
have an inhibitory effect on A
production(10) , the lack
of specificity of such compounds precluded a clear identification of
cellular compartments that contributed to A
production. Since the
vacuolar-type H
-ATPase(s) are thought to be
ubiquitously involved in the maintenance of the acidic milieu in
diverse intracellular acidic organelles(16) , we decided to
test bafilomycin A1 (baf A), a potent and selective inhibitor of the
vacuolar-type H
-ATPase(s) (17) as to its
effect on APP metabolism, including A
secretion.
293 cells (transformed human embryonic kidney cells)
overexpressing either APP751 wild-type (APP-WT) or
APP751 (APP-NL) were grown to semiconfluence and
subsequently metabolically labeled in the absence or presence of 1
µM baf A. Human fetal cortical cultures were prepared as
described previously(2) . Cultures were grown at least 30 days in vitro and were subsequently metabolically labeled for 16 h
in the absence or presence of 1 µM baf A. Bafilomycin A1
was obtained from Dr. K. Altendorf (Osnabrück,
Germany). A
was immunoprecipitated from aliquots of conditioned
medium (CM) using R1282 antiserum (obtained from Dr. D. Selkoe (Brigham
and Women's Hospital, Boston, MA)). Total s-APP was similarly
determined by immunoprecipitating aliquots of the CM with
-5(5) .
-s-APP was separately immunoprecipitated with
6C6(11) , and
-s-APP immunoprecipitated with 92 (wild-type
APP) (10) or SW192 (APP-NL) antibodies. The SW192 polyclonal
was produced essentially according to the method described for the
wild-type 92 antibody(11) , with the exception that the
synthetic pentapeptide antigen ended with NL rather than KM.
Cell-associated forms of APP were immunoprecipitated from aliquots of
the cell lysate protein with
-6(5) .
Metabolic labeling
and immunoprecipitations were carried out as described
before(5) . Labeling was carried out for the indicated period
of time with 100 µCi/ml L-[S]methionine in methionine-free
medium containing 10% heat-inactivated, dialyzed fetal bovine serum.
After labeling, CM were gently removed and the cells lysed in 1 ml of
lysis buffer (50 mM Tris pH 8.0, 0.15 M NaCl, 20
mM Na-EDTA, 1% sodium deoxycholate, 1% Triton X-100, and 0.1%
SDS) and the crude lysates were cleared of debris by centrifugation at
100,000
g and 4 °C for 5 min. Approximately 100
µg of total cell lysate or the equivalent amount of supernatant
were immunoprecipitated with 5 µl of antiserum or 5 µg of
antibody and 10 µl of protein A-Sepharose beads (Pharmacia Biotech
Inc.). Immunoprecipitated protein was solubilized from the beads after
boiling in reducing Laemmli buffer. Solubilized protein was separated
by SDS-polyacrylamide gel electrophoresis on 10-20% Tricine
gradient gels or 6% Tris-glycine gels. The gels were fixed, soaked in
Amplify (Amersham), dried, and exposed to Kodak X-AR autoradiography
film.
Treatment of cells stably transfected with SW-APP with baf A
at 1 µM leads to a virtually complete inhibition of A
release (-82%) from the transfected cells (Fig. 1; Table 1) as determined by the loss of the
4-kDa A
band,
immunoprecipitable by R1282. In contrast, there appears to be a
stimulation of secretion of the 3-kDa APP-derived fragment
(+234%), previously determined to start at either Leu-17 or
Val-18(18) . This fragment is presumed to derive from the
cell-associated
9-10-kDa APP carboxyl-terminal fragment
generated as a result of
-secretory cleavage.
Figure 1:
Immunoprecipitation of A from
conditioned medium of bafilomycin A1-treated 293 cells. 293 cells
(transformed human embryonic kidney cells) overexpressing APP751
wild-type (WT-APP) or SW-APP751 (APP-NL) were grown
to semiconfluence and subsequently metabolically labeled with
[
S]methionine for 4 h in the absence or presence
of 1 µM bafilomycin A1. Aliquots of supernatants
equivalent to 100 µg of cell lysate protein were immunoprecipitated
with 5 µl of R1282 antiserum.
Immunoprecipitation of s-APP from the CM with two separate APP
antibodies revealed that although total s-APP production was somewhat
diminished (-9%) from the SW APP-transfected cells (Fig. 2A; Table 1), there was actually a modest
increase in -s-APP secretion (+11%) (Fig. 2B; Table 1). In contrast, there is a dramatic inhibition of
-s-APP release (-55%) (Fig. 2C; Table 1) in
the presence of baf A. The less dramatic inhibition of total s-APP
appears to result as a consequence of this as well, since, on careful
examination of the immunoprecipitated total s-APP, which resolves into
a closely spaced doublet, it appears that bafilomycin treatment
abolishes the shorter (
-s-APP) component, accompanied by an
increase in intensity of the longer (
-s-APP) species (data not
shown). Treatment with baf A thus does not inhibit
-s-APP, but
selectively inhibits
-s-APP secretion, along with A
secretion.
Figure 2:
A, immunoprecipitation of total
s-APP from conditioned medium of baf A-treated 293 cells. 293 cells
overexpressing APP-WT or APP-NL were metabolically labeled for 4 h in
the presence or absence of 1 µM bafilomycin A1. Aliquots
of CM equivalent to 100 µg of cell lysate protein were
immunoprecipitated with 5 µg of 5 antibody, which specifically
recognizes total secreted s-APP. B, immunoprecipitation of
-s-APP from CM of baf A-treated 293 cells. CM aliquots as in A were immunoprecipitated with 5 µg of 6C6 antibody, which
specifically recognizes
-secretase-cleaved s-APP (
-s-APP). C, immunoprecipitation of
-s-APP from CM of baf A-treated
293 cells. CM aliquots as in A from 293 cells overexpressing
APP-WT were immunoprecipitated with 5 µg of 92 antibody, which
specifically recognizes
-secretase-cleaved s-APP wild-type
(
-s-APP-WT). In parallel, supernatants of 293 cells overexpressing
APP-NL were immunoprecipitated with 5 µg of SW192 antibody, which
specifically recognizes
-secretase-cleaved s-APP NL
(
-s-APP-NL).
Investigation of the fate of full-length APP in the cell lysates revealed that there is a selective increase in the mature APP full-length protein, but no detectable change in the immature holoprotein (Fig. 3). The mobilities of either species are not altered on SDS-polyacrylamide gel electrophoresis, suggesting that the effects observed with baf A are probably not owing to effects on protein glycosylation, for example.
Figure 3:
Immunoprecipitation of cell associated
full-length forms of APP from baf A-treated cells. 293 cells
overexpressing APP WT or APP751 NL were metabolically labeled for 4 h
as described before, and 100 µg of cell lysate protein were
immunoprecipitated with 5 µg of 6 antibody, which recognizes
both full-length mature APP (m-APP) and immature APP (i-APP).
Surprisingly, baf A failed to
inhibit A production from the WT APP (Fig. 1), expressed in
the same cell type. In fact, increased A
production (+48%)
was actually observed (Table 1). Treatment with baf A, however,
stimulated
-s-APP production (+125%) (Fig. 2, A and B; Table 1) and increased levels of mature
holoprotein (Fig. 3) detected in the cell lysates. These results
were similar to that observed with the SW APP. The lack of inhibition
of A
was paralleled by an undiminished secretion of WT
-s-APP
(+100%) (Fig. 2C). A slightly decreased
electrophoretic mobility of the
-s-APP also indicated baf
A-induced alterations in post-translational processing events.
The
differential effect of baf A on A production from the mutated APP
in comparison to the WT APP prompted us to investigate the effect of
the drug on a separate pathogenetic APP mutation, that at codon 717 (19) . As with WT APP, treatment of 293 cells stably
transfected with APP695
with baf A did not lead to any
detectable inhibition of A
production (Fig. 4); again, as
with the WT APP, a small increase was seen. Unlike the Swedish
mutation, the cellular expression of this mutation does not lead to an
overproduction of the A
peptide. It has been reported, instead,
that the expression of this mutation leads to a selective increase in
the longer, 1-42 form of the A
peptide(20) , which
is known to be more prone to aggregation than the shorter 1-40
peptide. It thus appears that the baf A inhibitory effect observed with
the SW APP in these cells is specific to the SW mutation and is not
shared by the Hardy mutation.
Figure 4:
Immunoprecipitation of A from CM of
baf A-treated 293 cells and a human neuroglioma cell line. 293 cells
overexpressing APP751-WT, APP751-NL, APP695 V717I, and the human
neuroglioma cell line HS683 overexpressing APP695 were metabolically
labeled for 4 h in the absence or presence of 1 µM baf A.
CM aliquots equivalent to 100 µg of cell lysate protein were
immunoprecipitated with 5 µl of R1282
antiserum.
We were interested in knowing whether
the insensitivity to baf A treatment observed with A produced from
WT APP in 293 cells represented an universal phenomenon. To this end,
we evaluated a human neuroglioma-derived cell line, HS683, stably
transfected with APP695. Immunoprecipitation from the CM of this cell
line revealed very robust production of a
4-kDa A
band,
comparable to the level of secretion from 293 cells transfected with
the SW APP (Fig. 4). Treatment with baf A resulted in a
significant inhibition of production of the A
fragment, sparing
the 3-kDa fragment. Thus, the cell type apparently determines whether
or not A
is produced from APP in an acidic milieu.
To
investigate these puzzling observations further, HMBC, which also
secrete A as a result of endogenous APP processing, were treated
with 1 µM baf A. CM was collected overnight and evaluated
using an enzyme-linked immunosorbent assay that specifically detects
A
in the CM from these cells(2) . Greater than 75%
inhibition of A
production was observed in these cultures (Table 2). As in the 293 cells expressing the Swedish mutation,
this was also accompanied by a dramatic inhibition of the
-s-APP,
whereas no significant effect was observed with
-s-APP. At the
levels of baf A tested, no cytotoxicity was evident. Removal of the
drug, in fact, led to a reversal of the inhibitory effect over a 48-h
recovery period (data not shown). Immunoprecipitation with R1282 from
the CM of HMBC treated with baf A for 24 h confirmed the selective
inhibition of release of the 4-kDa A
peptide (Fig. 5),
without loss of the 3-kDa fragment. Therefore, as in the 293 cells
expressing the SW APP, and the HS683 cells expressing WT APP, A
production from the primary cultures expressing genomic WT APP is thus
effectively inhibited by baf A as well.
Figure 5:
Immunoprecipitation of A from CM of
human mixed brain cultures in the absence or presence of baf A. Human
fetal cortical cultures were grown 30 days in vitro and were
subsequently metabolically labeled for 16 h in the absence or presence
of 1 µM baf A. Aliquots of CM equivalent to 100 µg of
cell lysate protein were immunoprecipitated with 5 µl of R1282
antiserum.
As selective and potent
inhibitors of the V-ATPases, the bafilomycins are capable of inhibiting
the acidification of diverse intracellular vesicles. Indeed, baf A has
been shown in a number of different studies to have dramatic effects on
specific cellular processes, such as phagosomal acidification,
lysosomal acidification and intracellular protein degradation,
endosomal carrier vesicle formation, and synaptic vesicle catecholamine
uptake(21, 22, 23, 24) . The
susceptibility to baf A has been used as diagnostic for the
rate-limiting involvement of the V-ATPases in these various processes.
The experimental observations in this report therefore strongly suggest
that an intracellular acidic compartment plays a critical role in the
generation and secretion of A from SW APP, but not WT APP, in 293
cells. The observed increased production of A
with the Swedish APP
may, at least in part, be due to the mutant protein being
differentially trafficked into an acidic intracellular compartment in
the 293 cells, much more conducive to both
-secretase cleavage and
release of A
. Differential intracellular trafficking of the SW APP
metabolites has been recently suggested to occur in polarized
cells(25) . By this analogy, increased trafficking of APP into
more acidic compartments in HMBC and the neuroglioma cell line may
explain the sensitivity to baf A inhibition of A
release. An
alternative explanation might be that the SW-APP is much more prone to
-secretase cleavage in an acidic compartment, and the increase in
pH as a consequence of baf A treatment thus has a more dramatic
inhibitory effect on this mutation, but not on WT. However, one would
then have to additionally posit that the
-secretase protease has
different pH optima toward SW as compared to the WT APP. In the absence
of an isolated
-secretase enzymatic activity, it may not be
possible to choose between these separate explanations.
These
observations, additionally, also directly link A production to the
occurrence of the
-secretase cleavage, reflected in the release of
-s-APP. In both the SW APP-transfected 293 cells, as well as in
the HMBC, the inhibition of A
release by baf A also strongly
inhibited the release of the
-s-APP as well. The lack of any
negative effect of the drug on either
-s-APP secretion or the
production of the 3-kDa APP fragment in all of the cell types tested
also suggests that
-secretase activity, in contrast to
-secretase, does not require an acidic environment. In one study (26) , treatment with 500 nM baf A led to the
inhibition of maturation and targeting of two separate lysosomal
enzymes, processes known to involve acidic compartments such as the
endosome and lysosome; yet, no effect on the concurrent processing and
secretion of either proalbumin, or proC3 was detected. The latter
events, mediated presumably by the constitutive secretory pathway
involving the trans-Golgi network, appear to be insensitive to
alterations of intravesicular pH. The insensitivity of
-secretase
activity toward baf A also confirms previous conclusions (15) regarding the likely cellular locale for this constitutive
secretory cleavage.
In both SW and WT APP-transfected 293 cells,
treatment with baf A is accompanied by a significant increase in
-s-APP (Fig. 2), as well as an increase in the level of
cell-associated mature holoprotein (Fig. 3). Lysosomal
degradation of APP has been suggested previously to be an important
component of total APP metabolism in 293 cells expressing WT
APP(8) . The inhibition of such degradative processes by baf A
may lead to the recycling of both WT and SW APP holoprotein into the
constitutive secretory pathway. Contrary to its effect on the 293
cells, baf A effected only relatively modest changes on either mature
holoprotein levels or on
-secretase cleavage in either HMBC or the
HS683 cells (data not shown).
The identities of the processes
sensitive to bafilomycin treatment, apparently crucial to
A/
-s-APP formation, are not clear from these experiments. It
is also unclear whether the presumed elevation of intravesicular pH
that leads to the
-s-APP and A
release inhibition is owing to
a direct effect on the enzyme(s) involved, or owing to indirect
effects, mediated perhaps by inhibition of vesicle fusion events, for
example. In any case, the availability of compounds such as baf A
stimulate further experimental work aimed at pinpointing the cellular
compartments which mediate A
production in different cells.
Knowledge gained from such studies would lead to increased
understanding of the cellular enzymology involved in the formation of
A
, which would be in the critical path to designing effective
therapeutic agents for Alzheimer's disease.