(Received for publication, September 20, 1995)
From the
-Amyloid (
A) is a normal metabolic product of the
amyloid precursor protein (APP) that accumulates in senile plaques in
Alzheimer's disease. Cells that express the Swedish mutant APP
(Sw-APP) associated with early onset Alzheimer's disease
overproduce
A. In this report, we show that expression of Sw-APP
gives rise to cell-associated
A, which is not detected in cells
that express wild-type APP. Cell-associated
A is rapidly
generated, is trypsin-resistant, and is not derived from
A uptake,
indicating that it is generated from intracellular processing of
Sw-APP. Intracellular and secreted
A are produced with different
kinetics. The generation of intracellular
A is partially resistant
to monensin and a 20 °C temperature block but is completely
inhibited by brefeldin A, suggesting that it occurs in the Golgi
complex. Monensin, brefeldin A, and a 20 °C temperature block
almost completely inhibit
A secretion without causing increased
cellular retention of
A, suggesting that secreted
A is
generated in a post-Golgi compartment. These results suggest that the
metabolism of Sw-APP gives rise to intracellular and secreted forms of
A through distinct processing pathways. Pathological conditions
may therefore alter both the level and sites of accumulation of
A.
It remains to be determined whether the intracellular form of
A
plays a role in the formation of amyloid plaques.
The deposition of -amyloid (
A) (
)in senile
plaques is a pathological hallmark of Alzheimer's disease
(Glenner and Wong, 1984; Masters et al., 1985).
A is a
normal product of the metabolism of amyloid precursor protein (APP) in
the secretory pathway (Haass et al., 1992; Seubert et
al., 1992; Shoji et al., 1992; Busciglio et al.,
1993). Mutations in APP that are linked to autosomal dominant
inheritance of Alzheimer's disease have been found to alter the
production of
A. Mutation of the two amino acids proximal to the N
terminus of
A has been described in individuals from two Swedish
families that develop early onset Alzheimer's disease (Mullan et al., 1992). Expression of APP containing the Swedish
mutation (Sw-APP) in transfected cells increases the production of
A by about 5-fold, suggesting a causal link between altered APP
processing and the development of Alzheimer's disease (Citron et al., 1992; Cai et al., 1993). In this report, we
show that the Swedish APP mutation not only increases
A production
but also results in abnormal intracellular accumulation of
A. The
processing pathways that give rise to intracellular and secreted
A
can be distinguished by their differential kinetics and sensitivities
to metabolic inhibitors and temperature block.
COS cells were transiently transfected with wild-type
APP and APP
and Sw-APP
cDNAs
and metabolically labeled followed by immunoprecipitation of
A and
APP from the culture medium and cell lysate. Cells expressing Sw-APP
secreted about 10-fold higher levels of the 4-kDa
A peptide than
cells expressing wild-type APP
when normalized for APP (Fig. 1A), consistent with previous reports (Citron et al., 1992; Cai et al., 1993). Immunoprecipitation
of the detergent-soluble lysate of cells expressing Sw-APP revealed the
presence of a labeled 4-kDa peptide, which was not detected in cells
expressing wild-type APP
and APP
or in
mock-transfected cells (Fig. 1B). The 4-kDa peptide
immunoreactivity was abolished by preabsorption of the
A antibody
with synthetic
1-40 peptide (Fig. 1D) and
did not appear after immunoprecipitation with an antibody to the APP
C-terminal domain. When cells were incubated at 4 °C, the
generation of cell-associated
A was completely inhibited (Fig. 1C), suggesting that cell-associated
A is
produced by active APP processing and not as an artifact of proteolysis
during the immunoprecipitation protocol. Cell-associated
A was
recovered in the supernatant after high speed centrifugation of the
detergent-extracted lysate suggesting that it is soluble (data not
shown). These results suggest that the cell-associated 4-kDa peptide is
antigenically and physically similar to the secreted 4-kDa
A
peptide. The low level of cell-associated
A precluded sequence
analysis of the N terminus.
Figure 1:
Accumulation of cell-associated A
in cells expressing Sw-APP. A, secreted
A; B,
cell-associated
A. Transfected COS cells expressing
APP
, APP
, or Sw-APP
and
mock-transfected cells were metabolically labeled with
[
S]methionine for 16 h. The culture medium and
cell lysates were immunoprecipitated for
A as described under
``Experimental Procedures.'' C, temperature
dependence of intracellular
A generation. Sw-APP-expressing cells
were pulse-labeled at 37 °C for 10 min and then chased for 90 min
at either 4 or 25 °C. Incubation at 4 °C completely prevents
generation of intracellular (Cell) and secreted (Sec)
A. D, trypsin resistance of cell-associated
A.
Sw-APP-expressing cells were untreated (Cntrl) or incubated
with trypsin for 10 or 20 min at 37 °C, and the cell lysates were
immunoprecipitated with the
1-40 antibody. Preabsorption of
the antibody with synthetic
1-40 peptide (Preab)
abolishes immunoreactivity. E, cell-associated
A in
Sw-APP-expressing cells is not due to
A uptake from the medium.
The first three panels show cell-associated
A in Sw-APP
transfectants (Cntrl) and in non-transfected COS cells
incubated for 6 or 24 h with medium containing radiolabeled
A. The
last two panels show
A remaining in the conditioned
medium after 6- and 24-h incubations with non-transfected COS cells.
Radiolabeled
A is derived from the medium of metabolically labeled
Sw-APP transfectants.
To determine whether cell-associated
A in cells expressing Sw-APP is intracellular or associated with
the cell surface, cells were treated with trypsin prior to lysis and
immunoprecipitation. The level of cell-associated
A was only
slightly decreased by trypsinization (Fig. 1D),
suggesting that it is mostly intracellular. In some labeling
experiments, the 3-kDa peptide was detected in a cell-associated form.
In contrast to the 4-kDa peptide, the cell-associated 3-kDa peptide was
trypsin-sensitive (Fig. 1D), suggesting that it is
mostly associated with the plasma membrane. We then determined whether
intracellular
A could be due to uptake of
A from the medium.
Conditioned medium from Sw-APP-expressing cells containing high levels
of radiolabeled
A was incubated for 6 and 24 h with
non-transfected COS cells. The level of labeled
A in the medium
was only slightly decreased after this incubation period (Fig. 1E). A low level of radiolabeled
A was
detected in association with nontransfected cells after incubation with
radiolabeled
A but did not exceed 20% of the level of
cell-associated
A in Sw-APP-transfected cells (Fig. 1E). Thus, most of the intracellular
A
produced from Sw-APP is not derived by uptake of
A from the
medium. These results suggest that cell-associated
A is
intracellular and is predominantly generated from intracellular
processing of Sw-APP.
The kinetics of production of intracellular
and secreted A were examined by pulse-chase labeling of HEK 293
cells stably transfected with Sw-APP. Intracellular and secreted
A
both appeared by 30 min of chase time (Fig. 2, A and B). Intracellular
A continued to accumulate after 45- and
60-min chase times and then declined by 120 min. In contrast,
A in
the medium continued to accumulate up to 120-min chase time (Fig. 2, A and B). Intracellular
A
reached a maximum of about 15% of secreted
A at 60-min chase time.
We also examined the time course of appearance of the 11.5- and 9-kDa
C-terminal fragments of APP, which are metabolic intermediates in the
processing of APP to
A and the 3-kDa peptide, respectively
(Busciglio et al., 1993; Haass et al., 1993; Higaki et al., 1995). The 11.5-kDa fragment appeared by 15-min chase
time, which slightly preceded the appearance of intracellular and
secreted
A (Fig. 2, A and B). In
contrast, the
-secretase-generated 9-kDa C-terminal fragment
accumulated more slowly, reaching a maximal level after 90-min chase
time (data not shown). The differential kinetics of production of
intracellular and secreted
A suggest that they are processed
differently and provide further evidence that intracellular
A is
not a contaminant of extracellular
A or a product of
A
endocytosis.
Figure 2:
Pulse-chase kinetic analysis of Sw-APP
metabolism to secreted and intracellular A and C-terminal APP
fragments. A, intracellular (IC) and secreted (SEC) 4-kDa
A and the 11.5-kDa C-terminal APP fragment (CTD) after 20 min of pulse labeling followed by chase in
unlabeled medium for the indicated times. Shown are fluorographs of 10%
Tris-Tricine (IC) or 13% Tris-Tricine (SEC) SDS gels. B, quantitative pulse-chase analysis of the appearance of
intracellular (
) and secreted
A (
) and the 11.5-kDa
C-terminal fragment (
). Values are the mean ± S.E.; n = 3. A and B are from separate
experiments.
To determine the sites of generation of intracellular
A from Sw-APP, we examined the effects of inhibitors of processing
and transport in the secretory pathway. Pretreatment of cells with
brefeldin A causes resorption of the proximal Golgi into the
endoplasmic reticulum and inhibits anterograde transport and maturation
of APP in the secretory pathway (Caporaso et al., 1992;
Gabuzda et al., 1994). Brefeldin A completely inhibited the
production of both intracellular and secreted
A from Sw-APP,
suggesting that these processing steps are unlikely to occur in the
endoplasmic reticulum or proximal Golgi (Fig. 3A). We
also examined the effects of the ionophore monensin, which inhibits the
maturation of newly synthesized proteins at the trans-Golgi and their
transport past the trans-Golgi network (Tartakoff, 1983). Incubation
with 7.5 µM monensin almost completely inhibited
A
secretion in both Sw-APP and wild-type APP-expressing cells but only
partially inhibited the generation of intracellular
A in cells
expressing Sw-APP (Fig. 3A). PhosphorImager analysis
showed that monensin inhibited
A secretion by 99 ± 0.2% but
inhibited the generation of intracellular
A by only 49 ±
5%. A monensin dose-response analysis showed that
A secretion was
almost completely inhibited by 2.5 µM monensin, whereas
intracellular
A production was only partially inhibited by 2.5
µM monensin, and no further inhibition was evident up to
10 µM monensin (Fig. 3B). Cell-associated
APP increased in monensin-treated cells, as previously reported
(Gabuzda et al., 1994). The differential sensitivity of
intracellular
A to monensin and brefeldin A is consistent with a
site of generation in the trans-Golgi. In contrast, the inhibition of
A secretion by monensin, without increased cellular retention of
A, is consistent with the generation of secreted
A in a
post-Golgi compartment.
Figure 3:
Effects of monensin, brefeldin A, and a 20
°C temperature block on the generation of intracellular and
secreted A. A, PhosphorImager scans of intracellular
A (IC) and secreted
A (SEC) in COS cells
expressing wild-type or Swedish mutant APP under control conditions (CNTL) and after treatment with 3 µg/ml brefeldin A (BFA) or 7.5 µM monensin (MON). B, monensin dose response of intracellular and secreted
A. Note that monensin almost completely inhibits secretion of
A but only partially inhibits generation of intracellular
A.
Values are expressed as percent of IC
A in the absence of monensin
(control) and represent the mean ± S.E. of three independent
experiments. C, incubation at 20 °C almost completely
inhibits generation of secreted
A but only partially inhibits
generation of intracellular
A. Values are molar ratios of
A:APP normalized to the 37 °C value and represent the mean
± S.E., n = 3. *, p < 0.01 relative
to control by ANOVA.
To further assess the site of intracellular
A generation, we examined the effects of a 20 °C temperature
block, which results in protein retention in the trans-Golgi (Matlin
and Simons, 1983). APP synthesis occurred at 20 °C but was
significantly reduced (data not shown). Hence, determinations of
A
were normalized for the level of APP. The 20 °C temperature block
almost completely inhibited
A secretion but only partially
inhibited the generation of intracellular
A (Fig. 3C). The level of intracellular
A generated
at 20 °C was similar to that observed in the presence of monensin (Fig. 3, B and C). These results provide
additional evidence that intracellular and secreted
A are
generated at distinct sites in Golgi and post-Golgi compartments,
respectively.
These experiments suggest that APP harboring the Swedish
mutation is processed to A at an early step in the secretory
pathway giving rise to a stable intracellular pool of
A. Hence,
the Swedish mutation results in both increased secretion and
intracellular accumulation of
A. Although intracellular
A is
a small fraction of the total
A produced, it may nevertheless play
a potentially important pathogenic role in plaque formation. The
appearance of
A in a cell-associated form has also been observed
in a neuronal cell line (Wertkin et al., 1993). Although we
have not observed intracellular
A in transfected cells that
overexpress wild-type APP, we cannot exclude the possibility that there
is a small pool that is below the limits of resolution. Nevertheless,
our results demonstrate that the Swedish mutant APP gives rise to
significantly increased accumulation of intracellular
A.
Several lines of evidence suggest that the intracellular and
secreted forms of A arise through distinct processing pathways.
First, the kinetics of generation of intracellular and secreted
A
are different. Second, the generation of intracellular
A is
inhibited by brefeldin A but is partially resistant to monensin and a
20 °C temperature block, suggesting that it occurs in the Golgi
complex, most likely the trans-Golgi. In contrast, both monensin and a
20 °C temperature almost completely inhibit the secretion of
A
without increasing cellular retention of
A. Thus, secreted
A
is generated in a post-Golgi compartment, which is distinct from the
Golgi site of generation of intracellular
A. These findings are
consistent with previous reports, which suggest that secreted
A is
produced in a post-Golgi compartment (Busciglio et al., 1993;
Haass et al., 1993; Higaki et al., 1995; Yamazaki et al., 1995), and are also consistent with the recent
observation that the cellular site of
-secretase cleavage is
altered in MDCK cells expressing Sw-APP (Lo et al., 1994).
The effects of the Swedish mutation on APP metabolism suggest that
inherited mutations alter not only the level but also the sites of
accumulation of A. Increased
A production has also been
demonstrated to result from aberrant APPs produced by deletion
mutations, incorrect APP isoform expression, and APP overexpression
(Zhong et al., 1994). Furthermore, we have previously
demonstrated that inhibition of energy metabolism markedly increases
the generation and intracellular accumulation of potentially
amyloidogenic C-terminal fragments of APP (Gabuzda et al.,
1994). Thus, a variety of genetic and non-genetic pathological
situations can alter the processing of APP and affect both the level
and cellular sites of accumulation of
A. It remains to be
determined whether intracellular accumulation of
A predisposes to
A aggregation and plays a role in the formation of amyloid
plaques.