(Received for publication, October 27, 1994; and in revised form, December 29, 1994)
From the
We analyzed the effect of the vacuolar H-ATPase
inhibitor bafilomycin A1 (bafA1) on the processing of
-amyloid
precursor protein (
APP). In kidney 293 cells stably transfected
with the wild-type
APP cDNA, bafA1 caused a stabilization of
mature
APP and its 10-kDa COOH-terminal fragment. Moreover, it
caused a 2-3-fold increase in secretion of soluble APP and
amyloid-
protein (A
). Interestingly, bafA1 treatment of cells
transfected with a mutant
APP isoform that occurs in a Swedish
kindred with familial Alzheimer's disease resulted in a decrease
of A
production and no increase of soluble APP secretion.
Identical results were obtained when the effect of bafA1 was analyzed
on fibroblasts derived from affected versus unaffected members
of the Swedish family. These data demonstrate a differential effect of
bafA1 on the production of A
derived from wild-type or Swedish
mutant
APP. Radiosequencing of A
derived from bafA1-treated
cells expressing wild-type
APP revealed a marked increase of
A
peptides starting at amino acids phenylalanine 4 and valine
-3 and a relative decrease of A
molecules beginning at the
typical NH
terminus of aspartate 1. Cells transfected with
the Swedish mutation and treated with bafA1 did not produce these
alternative A
peptides, so that bafA1 treatment resulted in a
decrease of A
starting at aspartate 1. Our data indicate that
multiple proteases are able to cleave A
at or near its NH
terminus. Inhibition of the protease cleaving at aspartate 1 by
bafA1 and perhaps other similar agents can result in an increase of
alternatively cleaved peptides.
One of the most characteristic features of Alzheimer's
disease (AD) ()is the accumulation of extracellular amyloid
plaques in the cerebral and limbic cortices and biochemically similar
deposits in selected cerebral blood vessels. Amyloid plaques are
predominantly composed of the 4-kDa amyloid
-peptide (A
).
A
is derived from a high molecular weight precursor protein, the
-amyloid precursor protein (
APP) by proteolysis (Kang et
al., 1987).
APP can apparently be cleaved by multiple
proteases to yield a variety of secreted derivatives (reviewed by Haass
and Selkoe, 1993). The predominant cleavage is mediated by an unknown
protease called
-secretase, leading to the secretion of the
NH
-terminal ectodomain of
APP, called soluble APP
(APP
) (Weidemann et al., 1989) and the retention
of a 10-kDa COOH-terminal fragment in the cell membrane (Selkoe et
al., 1988). Because this cleavage occurs within the A
domain
(at position 17 of A
) (Esch et al., 1990; Wang et
al., 1992), it prevents the production of A
. This cleavage
occurs in part on the cell surface (Sisodia et al., 1992;
Haass et al., 1992a, 1994a), although some studies have also
demonstrated apparent intracellular cleavage in an unknown subcellular
location (Sambamurti et al., 1992; DeStrooper et al.,
1993; Haass et al., 1995).
Besides the -secretory
pathway, a second trafficking pathway has been documented for
APP.
APP has been detected within clathrin-coated vesicles (Nordstedt et al., 1993) and shown to be reinternalized from the cell
surface as a full-length uncleaved molecule (Haass et al.,
1992a; Koo and Squazzo, 1994).
APP can subsequently be detected
within endosomes and lysosomes (Cole et al., 1989; Golde et al., 1992; Haass et al., 1992a; Estus et
al., 1992; Carporaso et al., 1994). However, it is not
clear whether
APP reaches endosomes only by reinternalization from
the cell-surface or also via a direct trafficking pathway from the
trans-Golgi network.
Besides the -secretase cleavage of
APP, an alternative proteolytic mechanism has been observed which
leads to the physiologic production and secretion of A
by cells in
culture (Haass et al., 1992b; Seubert et al., 1992;
Shoji et al., 1992; Busciglio et al., 1993). Two
enzyme activities have been postulated for A
generation (for
review, see Haass and Selkoe, 1993). Enzymes referred to as
- and
-secretases cleave at the NH
terminus (at a
methionine-aspartate bond) and the COOH terminus of A
(around
residues 40-42), respectively. These cleavages result in the
release of A
into the culture medium. An acidic intracellular
environment appears necessary for A
production (Shoji et
al., 1992; Haass et al., 1993; Koo and Squazzo, 1994).
Inhibition of
APP reinternalization leads to decreased A
generation (Koo and Squazzo, 1994). Such a mechanism is consistent with
the detection of COOH-terminal fragments of
APP that contain the
complete A
domain within isolated endosomes/lysosomes (Golde et al., 1992; Haass et al., 1992b; Nordstedt et
al., 1993).
Interestingly, missense mutations associated with
familial AD (FAD) have been mapped to the APP gene (for review,
see Hardy, 1992). The mutations are localized at the NH
terminus (Mullan et al., 1992), in the middle (Hendriks et al., 1992) or at the COOH terminus (Goate et al.,
1991) of the A
domain, close to the
,
, and
-secretase sites, respectively. Each of these mutations has now
been shown to strongly influence A
production. The double mutation
at the A
NH
terminus causes a 3-6-fold increase
in A
production (Citron et al., 1992, 1994; Cai et
al., 1993). The mutations at the A
COOH terminus result in
alternative
-secretase cleavages leading to COOH terminally
elongated peptides (Suzuki et al., 1994) that are believed to
aggregate faster than do wild-type peptides (Jarrett and Lansbury,
1993). The mutation in the middle of A
results in a relative
increase of A
peptides beginning at aspartate 1 and the increased
production of NH
terminally truncated A
-like peptides
(Haass et al., 1994b).
Little is known about the proteases
involved in A generation.
-Secretase cleaving at the
methionine-aspartate bond has been shown to be highly sequence specific
(Citron et al., 1995). Alternative NH
-terminal
cleavages of A
(Busciglio et al., 1993; Haass et
al., 1992b, 1993, 1994a, 1994b; Shoji et al., 1992)
result in the secretion of A
-like peptides not beginning at the
principal NH
terminus (aspartate). This finding raises the
question of whether the other peptides are generated by one and the
same
-secretase due to some degree of miscleavage or by multiple
-secretase-like enzymes with different sequence specificities and
subcellular localizations. To examine the latter possibility, we
attempted to find pharmacological agents that inhibit the predominant
methionine-aspartate cleavage activity but not the alternative
A
-like cleavages. Based on the knowledge that agents which
interfere with intracellular pH, such as NH
Cl and
chloroquine, inhibit A
production (Shoji et al., 1992;
Haass et al., 1993; Koo and Squazzo, 1994), we studied the
effects of bafilomycin A1 (bafA1), a more specific compound that does
not perturb the formation of intracellular organelles like Golgi
vesicles and endosomes. BafA1 is a macrolide antibiotic shown to be a
specific inhibitor of the vacuolar class of H
-ATPases in vitro (Bowman et al., 1988; Hanada et
al., 1990). In vivo, bafA1 can inhibit acidification of
endosomes, lysosomes, and phagosomes, resulting in the inhibition of
lysosomal protein degradation (Umata et al., 1990; Lukacs et al., 1992; Yoshimori et al., 1991). BafA1 also
inhibits acidification of the trans-Golgi network, which can result in
the inhibition of prohormone processing (Xu and Shields, 1994). In
addition it cannot be excluded that bafA1 might also inhibit
acidification of secretory vesicles (Xu and Shields, 1994). However,
bafA1 does not alter the morphology of vacuolar compartments, as do
weak bases and ionophores (Umata et al., 1990; Yoshimori et al., 1991; Johnson et al., 1993). Moreover, it has
been shown that bafA1 slows recycling of the transferrin receptor
without influencing its reinternalization (Johnson et al.,
1993).
Figure 1:
The
influence of bafA1 on the processing of APP. Kidney 293 cells
expressing the wild-type
APP
cDNA were metabolically
labeled for 2 h and chased for 2 h in the presence of 0.25 µM bafA1 or the Me
SO vehicle alone as a control. A, immunoprecipitation of A
from conditioned media with
antibody 1280. B, quantitation of the observed increase of
A
upon bafA1 treatment. Data are expressed as percent release
(untreated control = 100%) plus standard errors of the means
(± S.E.); n = 4. C, immunoprecipitation
of APP
from conditioned media with antibody B5. The lower band is the transfected
APP
isoform
of
APP (arrowhead). The upper band is the
endogenously expressed
APP
isoform. D,
quantitation of the observed increase of APP
upon bafA1
treatment. Data are expressed as percent release (untreated control
= 100%) ± S.E.; n = 4. E,
immunoprecipitation of membrane-associated
APP from cell lysates
with antibody C7. The upper band is mature N`- and O`-glycosylated
APP (N`/O`), and the lower
band is the immature N`-glycosylated
APP (N`). Arrow indicates the characteristic
COOH-terminal 10-kDa fragment generated by
-secretase cleavage. Asterisk indicates nonspecifically immunoprecipitated
protein.
The increased secretion of A and APP
is
dose-dependent. Elevated A
release was observed at bafA1
concentrations as low as 25 nM (Fig. 2A). The
effect of bafA1 on APP
secretion was also found to be
dose-dependent. However, we observed an increase of APP
secretion at a concentration of 2.5 nM of bafA1 (Fig. 2B). The observed difference in the
dose-dependent secretion of APP
and A
might indicate
that these proteolytic products of
APP are generated within
distinct subcellular compartments which are differentially affected by
bafA1. In agreement with published data on the action of bafA1 (Bowman et al., 1988; Hanada et al., 1990), our results
suggest a highly specific dose effect of bafA1 on the processing of
APP.
Figure 2:
The
effect of bafA1 in enhancing secretion of A and APP
is
dose-dependent. Kidney 293 cells expressing the wild-type
APP 695
cDNA were metabolically labeled for 2 h and chased in the presence of
the indicated concentrations of bafA1. A, A
and p3 were
immunoprecipitated from conditioned media with antibody 1280. B, APP
was immunoprecipitated with antibody
B5.
Figure 3:
The
kinetics of APP and A
release in pulse-chase
experiments. Kidney 293 cells expressing the
APP wild-type cDNA
were metabolically labeled for 15 min and chased for 2.5 h in the
presence of bafA1 or Me
SO as a control. The media were
completely exchanged after every 15-min time point. A,
APP
was immunoprecipitated with antibody B5. B,
A
and p3 were immunoprecipitated with antibody
1280.
Figure 4:
Detection of cell surface APP upon
bafA1 treatment. Kidney 293 cells expressing the wild-type
APP
cDNA were plated on poly-L-lysine coated glass coverslips.
After a 2-h treatment with either Me
SO (DMSO) as a
control (A) or 0.25 µM bafA1 (B), cells
were fixed with paraformaldehyde. Cell surface
APP was detected
with a mixture of the ectodomain monoclonal antibodies 5A3 and 1G7,
followed by a FITC-labeled goat anti-mouse secondary antibody. Note the
punctate cell surface expression of
APP upon bafA1 treatment.
Magnification
400.
Figure 5:
Treatment of kidney 293 cells expressing
the Swedish APP mutation with bafA1. Cells were metabolically
labeled for 2 h and chased for 2 h in the presence of 0.25 µM bafA1 or Me
SO as a control. A, conditioned
media were immunoprecipitated with antibody 1280. B,
quantitation of the immunoprecipitation shown in A. BafA1
causes a 30-40% reduction of A
secretion in cells expressing
the Swedish mutation, compared to the marked increase of A
secretion bafA1 causes in cells transfected with the wild-type
APP
cDNA. Data are expressed as percent increase/decrease over the
untreated control (± S.E.); n = 4. C,
APPs was immunoprecipitated with antibody B5. D, quantitation
of the immunoprecipitation shown in C (± S.E.); n = 4. BafA1 has no effect on the amount of APP
secretion. E, BafA1 stabilizes holoAPP and the 10-kDa
fragment in cells transfected with the Swedish mutation, as seen with
wild-type
APP (compare to Fig. 1E).
To show that this differential inhibition
of A production is not only observed in transfected 293 cells, we
repeated the same experiment with primary skin fibroblasts obtained
directly from affected and unaffected members of the Swedish FAD
family. It was recently shown by Citron et al.(1994) that
primary fibroblasts derived from these patients carrying the
APP
mutation show a striking overproduction of A
, similar to that
observed in transfected cell lines. Treating fibroblasts from
unaffected members of the Swedish pedigree with bafA1 again led to an
increase of A
secretion (Fig. 6A). In contrast,
fibroblasts cultured from patients with the
APP mutation secreted
lower amounts of A
in response to bafA1 treatment (Fig. 6A). Again, we observed a marked stabilization of
cell-associated
APP and the 10-kDa COOH-terminal fragment in
fibroblasts derived from both control subjects and patients with the
mutation (Fig. 6B).
Figure 6:
Treatment of primary skin fibroblasts
cultured from affected and unaffected members of the Swedish FAD family
with bafA1 or MeSO. A, A
was
immunoprecipitated from the media with antibody 1280. B,
membrane-associated
APP was immunoprecipitated from the cell
lysates with antibody C7 after the 2-h chase. Asterisk indicates nonspecifically precipitated
protein.
Figure 7:
A,
radiosequencing of A produced from kidney 293 cells expressing the
APP wild-type cDNA (open circles) and the
APP cDNA containing
the Swedish mutation (closed circles). Cells were labeled for 2 h with
2.5 mCi [
H] phenylalanine and chased for 2 h in
the presence of 0.25 µM bafA1. Data are expressed as %
counts per min (% CPM) per sequencing run (a total of 30 cycles is
shown). B, all of the observed NH
-termini of
A
peptides seen in both cell lines are shown as bars in reference
to the
APP amino acid sequence of the region -6 to 40 of
A
.
Very little is known about the identity and heterogeneity of
the secretases involved in the proteolytic processing of APP.
Sisodia (1992) suggested that
-secretase might be a membrane-bound
protease which cleaves its substrate in a sequence independent manner.
The cleavage specificity of
-secretase seemed to be mediated by
its cleaving at a fixed distance from the membrane and by the
recognition of an
-helical structure around the
-secretase
site. This model was supported by the observation that some mutations
close to the
-secretase cleavage site, which might diminish
-helical structure, inhibit cleavage by this enzyme, resulting in
the secretion of truncated APP
molecules (Haass et
al., 1994b).
In striking contrast to these properties of
-secretase,
-secretase cleaves at the methionine-aspartate
peptide bond in a highly sequence-dependent manner. In addition,
although
-secretase cleavage requires a membrane-bound substrate,
it does not require a fixed distance of the cleavage site from the
membrane (Citron et al., 1995).
-Secretase seems to be
localized in an acidic environment such as late Golgi vesicles or
endosomes, since alkalinizing agents such as NH
Cl,
monensin, and chloroquine reduce
-secretase activity (Shoji et
al., 1992; Haass et al., 1993; Koo and Squazzo, 1994).
As regards -secretase, it seems that its activity occurs
independently of a specific amino acid sequence at its cleavage site.
Substitution of various hydrophobic amino acids at position 42 of
A
does not noticeably change A
secretion. (
)However, FAD mutations at position 717 of
APP
(position 46 by A
numbering) cause an increased cleavage activity
of
-secretase at position 42 of A
(Suzuki et al.,
1994). Taken together, these data indicate that these secretases are
different types of enzymes with distinct biochemical properties and
perhaps different subcellular localizations.
In this paper, we
further characterized the biochemical properties of -secretase. We
focused on alternative NH
-terminal cleavages of A
resulting in the secretion of elongated or truncated A
peptides
(Busciglio et al., 1993; Haass et al., 1992b, 1993,
1994a, 1994b; Shoji et al., 1992). It is important to note
that these heterogenous peptides occur in the human brain and
cerebrospinal fluid (Gowing et al., 1994; Miller et
al., 1993; Seubert et al., 1992; Shoji et al.,
1992). Based on the knowledge that
-secretase is a highly
sequence-specific peptidase (Citron et al., 1995), we
hypothesized that different proteases are involved in the cleavage of
A
at the predominant methionine-aspartate peptide bond or at
alternative NH
-terminal cleavage sites. Here, we show that
bafA1 selectively reduces the formation of A
cleaved at the
methionine-aspartate peptide bond. In parallel, it causes a pronounced
increase of A
-like peptides starting at alternative NH
termini such as valine -3, phenylalanine 4, and isoleucine
-6. Interestingly, A
-like peptides with these alternative
NH
termini have also been observed without pharmacological
treatment. Madin-Darby canine kidney cells stably transfected with the
wild-type
APP cDNA secrete A
-like peptides beginning
predominantly at arginine 5 and valine -3 (Haass et al.,
1994a). Kidney 293 cells stably transfected with
APP cDNA
constructs containing COOH-terminal deletions or substitutions also
show a reduced secretion of peptides beginning at the
methionine-aspartate bond and produce increased amounts of A
beginning at valine -3, arginine 5, and phenylalanine 4 (Haass et al., 1993). (
)These data suggest that the
alternative cleavages of A
observed during bafA1 treatment may not
simply result from miscleavage by
-secretase due to a change in
its pH optimum. Rather, our current findings suggest that these
cleavages are mediated by different proteases with distinct biochemical
properties and/or different subcellular localizations. The latter
possibility is supported by the observation that bafA1 treatment causes
an increase of cell surface
APP (Fig. 4). Such an increase
of surface precursor is also observed when kidney 293 cells are
transfected with a variety of COOH-terminal deletion constructs. For
example, deleting the complete COOH-terminal cytoplasmic domain of
APP or mutating the cytoplasmic tyrosines at position 687 (within
the NPTY clathrin-coated pit motif) or at 653 cause an increase of cell
surface expression of
APP which is paralleled by the secretion of
increased amounts of A
peptides beginning at alternative NH
termini (Haass et al., 1993),
a result
analogous to that reported here after bafA1 treatment. However, this
hypothesis does not entirely rule out the possibility that
-secretase might have multiple cleavage activities at different pH
optima. Moreover, it should be noted that the the increased secretion
of A
after bafA1 treatment might only be observed in cell types
which are able to produce alternative NH
termini of A
.
In Chinese hamster ovary cells, which almost exclusively produce A
starting at aspartate 1, (
)bafA1 treatment does not result
in an increase of A
production. (
)
In this regard, it
is interesting to note that bafA1 causes a delayed increase of A
secretion (Fig. 3B). Together with our data on the
changes in A
NH
termini with bafA1, this finding leads
to the concept that A
starting at Asp
and the
A
-like peptides with alternative NH
termini are
secreted at different time points of the pulse-chase experiment. This
would lead to the hypothesis that alternative
-secretase-like
proteases are present in different subcellular environments at or close
to the cell surface. Inhibition of the
-secretase cleavage at the
``classical'' NH
terminus of A
(aspartate 1)
by bafA1 could result in an increased availability of substrate for
alternative
-secretases that are not inhibited by this agent. The
latter effect seems to be enhanced by the inhibition of intracellular
APP degradation by bafA1, thus leading to more
APP at or
close to the cell surface. The classical and alternative
-secretases seem to be expressed in varying amounts in different
cell types. For example, Madin-Darby canine kidney cells appear to
express lower amounts of the classical
-secretase, leading to the
secretion of peptides starting predominantly at alternative NH
termini; the aspartate 1 species represents only 10% of the total
secreted A
peptides in these cells (Haass et al., 1994).
Citron et al.(1994) have shown that kidney 293 cells
transfected with the Swedish mutation exclusively secrete A
peptides beginning at the characteristic aspartate 1 position. The same
cells showed a decrease of A
production upon bafA1 treatment (Fig. 5), whereas 293 cells expressing the wild-type
APP
cDNA secrete increased amounts of A
peptides. It is likely that
the Swedish mutation provides a preferred substrate for the classical
-secretase, in view of the fact that this mutation results in
3-6-fold increase of A
secretion in several cell types
(Citron et al., 1992, 1994; Cai et al., 1993) and
increases levels of the COOH terminally truncated form of APP
(Felsenstein et al., 1994). The fact that cells
transfected with the Swedish mutation produce less A
upon bafA1
treatment might therefore be explained by a sequence specific cleavage
by the alternative
-secretases. In the mutant Swedish
APP
molecule, the double mutation at the positions -1 and -2 of
A
might inhibit the recognition of the precursor by the
alternative
-secretases as well as enhancing recognition by the
classical
-secretase. Interestingly, bafA1 causes a preferential
increase of an A
-like peptide beginning at valine -3 in
wild-type
APP expressing cells, only 1 residue
NH
-terminal to the site of the Swedish mutations.
Therefore, these mutations might inhibit substrate recognition by the
protease which cleaves at valine -3, leading to an overall
reduced secretion of A
. In addition, A
derived from either
the mutant or wild-type genes may be generated in different subcellular
compartments, thus resulting in a differential effect of bafA1 on
A
generation. Such an interpretation would be supported by the
analysis of
APP trafficking in polarized cell types. In
Madin-Darby canine kidney cells, proteolytic products of wild-type
APP including APP
, A
, and p3 are secreted into
the basolateral compartment (Haass et al., 1994a; Haass et
al., 1995). However, in the case of the Swedish mutation,
truncated APP
generated by
-secretase is secreted into
the apical compartment (Lo et al., 1994). (
)This
result is consistent with the hypothesis that the cleavage of the
Swedish
APP substrate by
-secretase occurs in a different
compartment than the production of A
cleaved from wild-type
APP (Lo et al., 1994). Taken together, our data indicate
that a variety of biochemically distinct
-secretase-type proteases
are able to create the various NH
termini of A
peptides. These enzymes seem to be localized in different subcellular
compartments, with some alternative
-secretase enzymes close to or
at the cell surface, since bafA1 simultaneously increases cell surface
APP and enhances alternative
-secretase cleavage.
These
observations have important consequences for the generation of
pharmacological inhibitors of -secretases as potential drugs to
slow amyloid-plaque formation. For example, inhibition of the classical
-secretase (cleaving at the methionine-aspartate peptide bond) may
not result in a corresponding decrease of A
secretion. In at least
some cell types, A
-like peptides with alternative NH
termini may be secreted in elevated amounts. Some of these
peptides could have an enhanced ability to self-aggregate (Jarret and
Lansbury, 1993), resulting in no change or even an increase in amyloid
plaque formation.