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INTRODUCTION |
The PS11 protein is
thought to play an important role in Alzheimer's disease (AD).
Mutations in PS1 have been linked to familial Alzheimer's disease
(1-3). The mutations in PS1 and PS2 that are linked to familial AD
most likely cause AD by increasing production of A
1-42
(4, 5). The A1-42 peptide is a particularly hydrophobic
form of A that rapidly aggregates in solution (6). The increased
production of A1-42 seen in patients with PS1 or PS2
mutations is thought to cause familial AD by accelerating the
accumulation of A aggregates and the formation of neuritic plaques,
which are one of the principal pathologic components in AD.
A is generated by cleavage of its parent protein, amyloid precursor
protein (APP). During its processing, APP is alternatively cleaved to
generate either A or a secreted form of APP termed APPs
. The
cleavages that generate A occur at two positions. The putative
protease that cleaves APP at the C terminus of the A domain is
termed 
-secretase, whereas cleavage at the N terminus of the A
domain is carried out by a putative protease termed -secretase (7,
8). Cleavage by the putative protease, -secretase, occurs at a site
corresponding to A16 and generates a form of APP that is
normally secreted, termed APPs (9, 10). In addition, both pathways
generate C-terminal fragments of APP that are internalized and
degraded. Cleavage by secretase also generates APPs, which is
similar to APPs except that it lacks A1-16.
Secretion of APPs and A is tightly coupled to the activity of
signal transduction systems (11). Activation of protein kinase C (PKC)
or extracellularly regulated kinase (ERK) stimulates APPs secretion
and inhibits A secretion (12-14).
The mechanism through which mutations in PS1 alter APP processing is
poorly understood. Recent studies suggest that there is a direct
connection between APP processing and PS1. PS1 may form a complex with
APP in the endoplasmic reticulum (15). PS1 also appears to be required
for at least one step in the processing of APP. DeStrooper et
al. (16) recently showed that PS1 is required for -secretase
activity, which is a proteolytic cleavage that generates the C terminus
of the A (16). Naruse et al. (17) have confirmed this
observation, and Wolfe et al. (18) have shown that mutation
of PS1 can interrupt this process.
Although deficits in A production in cells lacking PS1 were observed
by both groups (16, 17), several important questions were raised by
these studies. Although APP processing is known to be regulated by
protein kinase C, neither group studied the effects of loss of PS1 on
the regulation of APP processing. In addition, neither group
investigated whether PS2, a close homologue of PS1, could compensate
for any of the defects associated with loss of PS1. Finally, DeStrooper
et al. (16) observed normal basal APP secretion in the cells
lacking PS1, whereas Naruse et al. (17) observed that cells
lacking PS1 secrete more APP than wild-type cells.
We have now investigated these questions. Consistent with the published
literature, we find that cells lacking PS1 secrete less A. In
addition, we find that cells lacking PS1 are unable to increase
secretion of APP following treatment with agents that stimulate PKC or
ERK. This occurs despite their ability to activate components of the
ERK cascade. We also observe that overexpression of PS2 can
compensate for the lack of PS1.
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MATERIALS AND METHODS |
Cell Lines, Antibodies, and Plasmids--
293 HEK cells were
grown in high glucose Dulbecco's modified Eagle's medium plus 10%
fetal bovine serum supplemented with 500 µg/ml G418, as needed.
Fibroblasts were grown in Dulbecco's modified Eagle's medium plus
10% calf serum. The antibodies used for the analyses include 22C11
(0.1 µg/ml, Roche Molecular Biochemicals, against the N terminus of
APP), 6E10 (1:1000, Senetek, against A1-16), CT15
(1:5000, against the C terminus of APP, provided by Eddie Koo,
University of California, San Diego), and PS1loop (1:1000,
provided by Sam Sisodia, University of Chicago). The PS1 and PS2
plasmids were provided by John Hardy (Mayo Clinic, Jacksonville, FL).
Alg3 was provided by Luciano D'Adamio (NIAID, National Institutes of
Health). The plasmids were cloned into pcDNA3 vectors.
Immunoblotting--
Cells were harvested with SDS lysis solution
(2% SDS, 10 mM Tris, pH 7.4, 2 mM -glycerol
phosphate, 1 µM 4-(2-aminoethyl)benzenesulfonyl fluoride). Protein was determined using the BCA assay (Pierce), and 30 µg per lane was run on 14% polyacrylamide gels and transferred to
nitrocellulose (200 mAmp, 6 h). The nitrocellulose was then incubated 1 h in 5% milk/phosphate-buffered saline, washed,
incubated overnight in 1° antibody, washed, and then incubated for
3 h in peroxidase-coupled 2° antibody and developed with
chemiluminescent reagent (DuPont).
Secreted APP--
Cells were plated at 106
cells/well in 35-mm dishes. The following day, the cells were washed
with serum-free medium, transferred to 1 ml of Opti-MEM (Life
Technologies, Inc.) without serum (as described below), and then
treated with 1 µM phorbol 12-myristate 13-acetate (PMA)
or 10 ng/ml epidermal growth factor (EGF) for 1 h. The medium was
then collected and centrifuged and 50 µl of heparin-agarose (Bio-Rad)
was added for a 1 h, 4 °C incubation in order to bind the APP.
The heparin agarose was pelleted, washed four times with
phosphate-buffered saline/0.1% Triton X-100, mixed with 25 µl of
Laemmli sample buffer, boiled for 5 min, and immunoblotted. The
resulting films were quantitated by video densitometry using the NIH
Image program.
A Enzyme-linked Immunosorbent Assay--
The assay was
performed as described previously (19). For total A measurement, we
used the anti-A1-16 antibody 3160 as the capture
antibody and peroxidase-coupled 4G8 as the detection antibody (19).
Assays specific to A40 were done by Christopher Eckman
as described by Suzuki et al. (19).
Transfections--
DNA was transfected into freshly plated cells
using LipofectAMINE Plus (Life Technologies, Inc.). Transfections were
performed in Opti-MEM (Life Technologies, Inc.) using a ratio of 2 µg
of DNA:6 µl of LipofectAMINE:9 µl of Plus reagent per 1 ml of
Opti-MEM. For detection of Elk, the Elk Pathdetect system (Stratagene)
was used. Luciferase measurements were made using a Turner luminometer.
Metabolic Labeling--
35-mm dishes containing 1 million cells
each were preincubated in methionine-free, serum-free medium for 30 min. The cells were then incubated with 250 µCi of
[35S]methionine in fresh methionine-free, serum-free
medium for 30 min and then washed and incubated in normal growth
medium. At the indicated times, the cells were lysed in lysis buffer
(phosphate-buffered saline, pH 7.4, 1% Nonidet P-40, protease
inhibitors), and the lysates were cleared by centrifugation. The
lysates were then incubated with CT15 (1:500) for 1 h, washed
three times in lysis buffer, and incubated with protein A (25 µl,
1 h). The conjugates were then centrifuged, washed three times in
lysis buffer, electrophoresed on 12% polyacrylamide gels, dried down,
and then exposed to film.
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RESULTS |
APP Secretion Is Reduced in Fibroblasts Lacking
PS1--
SV40-transformed fibroblasts were generated from E14 PS1
knockout mice (20, 21). Two types of cell lines were used, a line
hemizygous (+/
) for PS1 and a line nullizygous (/) for PS1. The
absence of PS1 in the nullizygous mice was assessed by polymerase chain
reaction (not shown) and confirmed by immunoblotting with an antibody
against PS1 using anti-PS1loop (Fig.
1A).
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Fig. 1.
A, expression of PS1 in transformed
fibroblasts from PS1 +/ and / mice. Total lysates were
immunoblotted using the anti-PS1loop antibody. PS1 was
absent in the / cell lines and appeared mainly as the 17-kDa
cleavage fragment and the +/ cell line (arrow).
B, immunoblot using antibody 22C11 of secreted APP from +/
and / cell lines following stimulation with 1 µM PMA
for 1 h. PMA did not increase secretion of APP in the / cell
line. C, quantitation of data immunoblot from B
using video densitometry. A statistically significant increase in APP
secretion was seen only with the +/ cells. *, p < 0.05. D, immunoblot using antibody 22C11 of secreted APP
from +/ and / cell lines following stimulation with 10 ng/ml EGF
for 1 h. EGF did not increase secretion of APP in the / cell
line.
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Next, we took the PS1 +/ and PS1 / lines and examined the
secretion of APP under basal conditions and after treatment with two
agents known to stimulate APP secretion by selectively activating secretase, PMA and EGF (22, 23). The fibroblast cell lines were
preincubated in serum-free medium, transferred to fresh serum-free medium, and treated with 1 µM PMA or 10 ng/ml EGF for
1 h. The secreted APP was precipitated and immunoblotted with
22C11, which is an antibody capable of detecting murine APP, as well as
the human APPs, APPs, and amyloid precursor-like proteins. The
PS1 +/ cells showed a 50 ± 4% (p < 0.05)
increase in APP secretion following stimulation with PMA, whereas the
PS1 / cells showed no increase (6 ± 14%) in secreted APP
(Fig. 1, B and C). Following treatment with EGF,
the PS1 +/ cells also showed a robust increase in APP secretion,
whereas the PS1 / cells showed no increase and instead showed a
reduction in APP secretion (Fig. 1D), suggesting an
impairment in the stimulation of APP secretion by EGF and PMA.
To determine whether APP secretion was also impaired in nontransformed
cells lacking PS1, we examined the APP secretion in primary embryonic
fibroblasts generated from E14 mice that were homozygous (+/+),
heterozygous (+/), or nullizygous (/) for PS1. The primary
embryonic fibroblasts were then plated at a density of 250,000 cells
per 35-mm dish, and the secretion of APP was examined under basal
conditions or following treatment for 1 h with 1 µM
PMA or 10 ng/ml EGF. The results paralleled those seen with the
transformed cell lines. PMA and EGF were able to increase APP secretion
in primary embryonic fibroblasts expressing PS1 but were unable to
increase APP secretion in primary embryonic fibroblasts lacking PS1
(Fig. 2, A and B).
The basal level of APP secretion was similar for all cells. Thus, both
primary cells and transformed lines lacking PS1 are unable to increase
APP secretion following stimulation with agents that activate PKC or
ERK.
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Fig. 2.
A, immunoblot using antibody 22C11 of
secreted APP from primary cultures of PS1 +/+, +/, and / murine
fibroblasts following stimulation with 10 ng/ml EGF for 1 h. EGF
did not increase secretion of APP in the / B,
quantitation of the absorbance of the APPs reactivity from the PS1 +/+,
+/, and / cells shows that EGF stimulates APP secretion only in
cells from PS1 expressing animals. *, p < 0.05;
n = 3 for each point. C, immunoblot using
the antibody 6E10 to detect secreted APPs from PS1 +/ and /
cell lines that had been transfected with varying amounts of a human
APP expression vector. Secretion of APP was reduced in the / cell
line. D, measurement of ERK/ELK activity using luciferase
constructs in PS1 +/ and / cell lines following stimulation with
10 ng/ml EGF for 24 h. *, p < 0.0001.
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To determine whether loss of PS1 specifically inhibited APPs
secretion, we transiently transfected the PS1 +/ and PS1 / lines
with varying amounts of a human APP expression construct driven by a
cytomegalovirus promoter. This allowed use of the antibody 6E10, which
recognizes the A1-16 sequence that is specific to human
APP and is present in APPs but absent from APPs and amyloid
precursor-like proteins. Two days later, we incubated the cells with
fresh medium for 1 h and precipitated and immunoblotted the
secreted human APP. Parallel transfections with a constitutively active
pGL3 luciferase reporter showed equal transfection efficiencies between
the two cell lines. The PS1 +/ fibroblasts transfected with human APP
showed dose-dependent increases in secreted human APP. The
PS1 / line showed a lower level of APP secretion at each dose and
reached a plateau at a total level of secreted human APP that was half
that of the PS1 +/ line, as determined by densitometry (Fig.
2C). These results suggest that fibroblasts lacking PS1 have
impaired secretion of APPs.
APP secretion has been shown to be regulated by the ERK cascade (22).
Absence of ERK signaling could also explain the inability of PS1 /
cells to increase APP secretion. Hence, we examined EGF signaling in
the PS1 / and PS1 +/ cell lines in order to determine whether the
ERK signaling cascade was functioning. To examine this question, we
tested the ability of EGF to activate Elk, a member of the MAP kinase
cascade. The PS1 +/ and PS1 / cell lines were transfected with
plasmids containing luciferase driven by a Gal4 upstream activating
site and a chimeric Elk1/Gal4 construct. The following day, the cells
were treated with 10 ng/ml EGF for 24 h, and then luciferase
activity was examined. EGF increased luciferase activity 4-6-fold
above basal levels in both PS1 +/ and PS1 / lines (Fig.
2D). These results indicate that EGF is capable of
activating Elk in the PS1 / cells. In contrast, EGF does not
stimulate APP secretion in PS1 / cells (Fig. 1D). These results indicate that loss of ERK/Elk signaling does not account for
the inability of EGF to stimulate APP secretion in PS1 / cells.
Transfection with PS1 Rescues the PS1 / Phenotype--
To
verify that the deficits in APP secretion from the PS1 / cells
resulted specifically from lack of PS1, we examined whether transfecting in PS1 restores normal secretion of APP. PS1 / cells
were transfected with the human APP expression vector (1 µg) and
either wild-type PS1 (1 µg) or a control plasmid (-galactosidase, 1 µg). Two days after transfection, the cells were preincubated in
serum-free medium for 2 h and then placed in fresh serum-free medium +/ 1 µM PMA for 1 h. The APP was
precipitated, and human APP was immunoblotted with the 6E10, which
recognizes human APP and therefore identifies only APP secreted from
the transfected cells. PS1 / cells transfected with PS1 increased
APP secretion during treatment with PMA (Fig.
3A, lane 2); in contrast, the PS1 / control cells showed no change in APP secretion in response to PMA treatment (Fig. 3A, lane 4). Because each point was
analyzed in triplicate, we also quantitated the results by densitometry in Fig. 3B. The ability of PS1 to restore normal patterns of
APP secretion indicates that the defect in APP secretion seen in the PS1 / cells results specifically from the lack of PS1
expression.
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Fig. 3.
Transfection of PS1 / cells with PS1
rescues APP secretion. A, PS1 / cells were
transfected with a human APP expression vector (1 µg/ml) and either 1 µg/ml of wild-type PS1 (lanes 1 and 2) or 1 µg/ml of control plasmid (-galactosidase) (lanes 3 and
4). Two days after transfection, the cells were preincubated
in serum-free medium for 2 h, transferred to fresh serum-free
medium, and incubated +/ 1 µM PMA for 1 h. The
secreted APP was isolated and immunoblotted with antibody 6E10. All
transfections were done in triplicate, and representative lanes are
shown. B, quantitation of the absorbance of the APPs band
for each of the lanes shows that cells transfected with PS1 exhibited a
68 ± 4% increase in APP secretion during PMA treatment, whereas
cells not expressing PS1 showed no increase in APP secretion during PMA
treatment. *, p < 0.05; n = 3 for each
condition. C, PS1/ cells were transfected with 2 µg/ml
of control plasmid (-galactiosidase) (lanes 1 and
2), or 2 µg/ml of wild-type PS1 (lanes 3 and
4). Two days after transfection, the cells were preincubated
in serum-free medium for 2 h and then transferred to fresh
serum-free medium and incubated with or without 1 µM PMA
for 1 h. The secreted APP was isolated and immunoblotted with
antibody 22C11. All transfections were done in triplicate, and
representative lanes are shown.
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To determine whether the effect of the rescue was directly related to
the presence or absence of PS1, rather than a result of overexpression
of both PS1 and APP, we determined whether transfecting with PS1
rescues endogenous APP secretion. PS1 / cells were transfected with
either wild-type PS1 (2 µg) or a control plasmid (-galactosidase,
2 µg). Two days after transfection, the cells were preincubated in
serum-free medium for 2 h and then placed in fresh serum-free
medium with or without 1 µM PMA for 1 h. The APP was
immunoprecipitated and immunoblotted using 22C11, which detects the
native mouse APP secretion. Transfecting PS1 / cells with human PS1
restored their ability to increase APP secretion after stimulation by
PMA (Fig. 3C, lanes 3 and 4). The ability of
transfected PS1 to restore normal secretion of endogenous APP confirms
that this is a specific function of PS1.
Cells Lacking PS1 Have Higher Levels of Cellular APP Cleavage
Products--
To determine whether APP secretion was reduced because
of less cleavage of cellular APP or less secretion of APP, we
immunoblotted cell lysates with antibodies to the N and C termini of
APP separately. The antibody 22C11 binds to the N terminus of APP and
therefore recognizes both holo-APP and APPs (both migrate at about 110 kDa during electrophoresis). In contrast, the antibody CT15 binds to
the C terminus of APP and recognizes holo-APP and the APP C-terminal fragment, but not APPs (the APP C-terminal fragment migrates at about
16 kDa during electrophoresis). To control for loading, we reprobed the
immunoblots with anti-actin antibody. Immunoblots with 22C11 showed
26% more APP/APPs reactivity in the lysates from PS1/ cells than
in the lysates from PS1 +/ cells after normalizing for loading by
quantitating staining with the anti-actin antibody (Fig.
4, A, upper panel, and
C). To investigate whether the increased reactivity in the
PS1 / cells resulted from increased levels of holo-APP or increased
levels of APPs, we probed immunoblots of cellular lysates with antibody
CT15, which does not detect APPs. Unlike the results seen with antibody
22C11, immunoblots with antibody CT15 did not show increased holo-APP
reactivity in the lysates from PS1 / cells compared with the
lysates from PS1 +/ cells (Fig. 4, B, upper panel, and
C). The unchanged (or slightly reduced) holo-APP reactivity
seen using the CT15 antibody contrasts with the increased reactivity
seen using the 22C11 antibody and suggests that in PS1 / cells some
of the holo-APP is cleaved, to yield APPs, but not secreted. The
cleaved cellular APPs would not be detected by CT15, but the cleaved
APP would be detected by 22C11, which also accounts for the increased
reactivity seen in PS1 / cells with 22C11. Consistent with
increased cleavage of APP in the PS1 / cells, we also observed that
the amount of APP C-terminal fragments were higher in the PS1 /
cell lysates (Fig. 4B, lower panel). The increased levels of
cellular APPs and APP C suggest that cleavage of APP is increased in
PS1 / cells, but that the APPs is not secreted.
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Fig. 4.
Analysis of cellular APP expression in PS1
/ and +/ cells. A, lysates from PS1 / and
+/ were immunoblotted with the anti-APP N-terminal antibody 22C11,
and then the immunoblots were reprobed with anti-actin antibody to
control for loading. The PS1 / cells showed more 22C11 reactivity
than the PS1 +/ cells. B, the same lysates were also with
anti-APP C-terminal antibody CT15, and then the immunoblots were
reprobed with anti-actin antibody. The APP C-terminal fragments
(arrow, CTF) showed more reactivity in PS1 / cells than
in PS1 +/ cells, but the holo-APP bands (arrow, holo-APP)
showed no increase in reactivity in the PS1 / cells. C,
quantitation of the density of the bands. The density of each anti-APP
band was determined and then normalized to the relative density of the
corresponding anti-actin band. **, p < 0.003; *,
p < 0.05; n = 3 per group.
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Next, we metabolically labeled the PS1 / and PS1 +/ cells to
compare the turnover times for the APP and C-terminal fragments. PS1
/ and +/ cells (106 cells/dish) were labeled with
35S methionine for 30 min and then incubated for 0, 30, or
60 min in normal serum-free medium not containing radioactivity, and CT15 was used for the immunoprecipitation. No difference was seen between the disappearance of holo-APP in the PS1 / cells
versus the PS1 +/ cells (Fig.
5A); however, APP C-terminal
fragments were more abundant at all time points in the PS1 /
lysates compared with PS1 +/ lysates (Fig. 5B). The
persistence of APP C-terminal fragments in the PS1 / cells suggests
that loss of PS1 impairs the catabolism of these fragments.
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Fig. 5.
Metabolic labeling of PS1 / and +/
cells with [35S]methionine. PS1 / or +/ cells
were pulsed with [35S]methionine for 30 min, and APP was
immunoprecipitated with CT15. Each lane was then quantitated by video
densitometry after subtracting the background for each lane. No
difference was seen between the PS1 +/ and / cell lines for the
metabolism of holo-APP (A), whereas C-terminal fragments
were more abundant at all time points in the PS1 / cells
(B).
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The Levels of APP C-terminal Fragments in 293 HEK Cells Are Also
Sensitive to the Amount PS1 Expression--
The increased levels of
APP C-terminal fragments seen in PS1 / cells indicates that loss of
PS1 increases the levels of APP C-terminal fragments. To determine
whether increasing expression of PS1 decreases levels of APP C-terminal
fragments we immunoblotted cell lysates from 293 HEK cells stably
transfected with wild-type PS1, FAD1 PS1, or control. Immunoblots with
an antibody against PS1, PS1loop, shows that the
transfected cells express much more PS1 than do the control cells (Fig.
6A). Immunoblots with the antibody CT15 showed that the levels of APP C-terminal fragments correlates with the levels of PS1. Cells stably transfected with wild-type PS1 showed lower levels of APP C-terminal fragments than
control cells (Fig. 6, B and C). Cells stably
transfected with FAD1 PS1 also showed lower levels of APP C-terminal
fragments than control cells (data not shown). The correlation between
APP C-terminal fragment levels and PS1 levels in 293 HEK cells shows that APP processing is affected by either increases or decreases in PS1
expression.
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Fig. 6.
Overexpression of PS1 reduces the levels of
APP C-terminal fragments. A, lysates from 293 HEK cells
stably transfected with vector, wild-type PS1, or FAD1 PS1 were
immunoblotted with anti-PS1 antibody PS1loop (lane
1, control; lane 2, wild-type PS1; lane 3, A146E PS1). Overexpression of PS1 is most evident in the amount of
holo-PS1 (upper arrow). The lower arrow indicates the
presence of the normal endoproteolytic product of PS1. B,
lysates from the same cell lines were immunoblotted with anti-APP
C-terminal antibody CT15. The immunoblots showed that overexpression of
wild-type or mutant PS1 reduced the levels of APP C-terminal fragments
(CTF, small arrow) but did not change the levels of holo-APP
(large arrow).
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A Secretion Is Reduced in Cells Lacking PS1--
The reduction
in -secretase cleavage of APP seen in cells lacking PS1 raises the
possibility that A secretion is reduced in cells that lack PS1. To
examine A secretion, we transfected PS1 +/ and PS1 / cells
with an expression vector coding for human APP, immunoblotted the cell
lysates with the human specific anti-APP antibody 6E10 to determine the
level of expression of human APP in the cells, and then measured A
secreted into the medium from the cells by enzyme-linked immunosorbent
assay. We were able to express human APP in both the PS1 +/ and /
cells (Fig. 7A). Enzyme-linked
immunosorbent assay measurements of A secretion from the cells over
a 24-h period showed that the cells expressing PS1 produced much more
A than the cells lacking PS1, even after normalizing for human APP
expression (Fig. 7B). Thus, loss of PS1 reduces A
secretion.
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Fig. 7.
PS1 / and +/ cells were transfected
with a human APP cDNA construct. Two days later, fresh medium
was placed on the cells, and the cells were incubated for an additional
24 h. A, the lysates were immunoblotted with 6E10, an
antibody that detects human APP, to measure the amount of expression of
human APP. The bands were then quantitated by densitometry.
B, the amount of A secreted from the cells analyzed in
A was quantitated by enzyme-linked immunosorbent assay
following normalization to the amount of human APP transfected. The
amount of A secreted from PS1 / was less than that secreted from
the PS1 +/ cells (p < 0.0001; n = 3).
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Transfection with PS2 Rescues the PS1 / Phenotype--
PS2 is
a close homologue of PS1 and has been shown to bind APP, like PS1 (24).
Whether PS2 also regulates APP processing is unknown. Cells lacking PS1
might be a useful system for investigating the role of PS2 because the
absence of PS1 eliminates the possibility of PS1 activity masking
potential actions of PS2. To investigate this issue, we examined the
effects of increasing PS2 expression in the PS1 / cells on APP
C-terminal fragments and secreted APP. PS1 / cells were transfected
with human APP (1 µg/ml) and either PS2 (1 µg/ml) or control
(-galactosidase, 1 µg/ml). Two days after the transfection, the
cells were preincubated in serum-free medium for 2 h and then
transferred to fresh serum-free medium with or without 1 µM PMA for 1 h. The lysates and secreted APP were
then collected and immunoblotted with CT15 and 6E10, respectively, to
monitor levels of APP C-terminal fragments and APPs. Cells transfected
with PS2 exhibited lower levels of APP C-terminal fragments and
secreted less APP under basal conditions (Fig.
8, A and B). The
levels of APP C-terminal fragments and APPs were both increased in the
PS2 transfected cells following treatment with PMA (Fig. 8,
A and B). The reduction in APP C-terminal
fragments and the restoration of PMA responsiveness in cells
transfected with PS2 indicates that PS2 can substitute for PS1 in
regulating APP processing. Thus, PS2 also has the ability to
participate in APP processing.
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Fig. 8.
Restoration of normal APP processing by
transfection with PS2. A and B, PS1 /
cells were transfected with PS2. Two days later, the cells were
preincubated in serum-free medium for 2 h, placed in fresh
serum-free medium, and treated with PMA or left untreated for 1 h.
The secreted APP was collected and immunoblotted with 22C11, and
lysates from the cells were immunoblotted with CT15. Transfection of
PS1 / cells with PS2 restores PMA responsiveness of APP secretion
(A) and reduces basal levels of APP C-terminal fragments
(B). C, transfection of PS1 / cells with
Alg3. Two days after transfection with Alg3, the cells were incubated
in serum-free medium for 2 h, and lysates of the cells were then
immunoblotted with CT15. Transfection of PS1 / cells with Alg3.
increases levels of APP C-terminal fragments (CTF, lower
arrow) but does not change levels of holo-APP (APP,
upper arrow).
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Previous studies indicate that PS2 promotes apoptosis and that Alg3, a
short C-terminal segment of PS2, inhibits apoptosis (25-27). The
opposing actions of PS2 and Alg3 suggests that Alg3 might inhibit PS2
function. The effects of Alg3 on APP processing, though, are unknown.
PS1 / cells constitute a good system for investigating the action
of Alg3 because the cells are unable to use PS1 to compensate for any
potential inhibition of PS2 activity. To investigate this question we
transfected PS1 / cells with Alg3 (2 µg) or control
(-galactosidase, 2 µg) and examined the expression of APP
C-terminal fragments. Cells transfected with Alg3 showed increased
levels of APP C-terminal fragments compared with control cells (Fig.
8C). This increase in APP C-terminal fragments contrasts
with the decrease in APP C-terminal fragments seen after transfection
with PS2. Thus, Alg3 and PS2 have opposing actions on APP C-terminal
fragments. We also examined the effects of Alg-3 on secreted APP and
found that Alg3 did not restore PMA responsiveness, unlike PS2 (data
not shown). These data suggest that Alg3 interferes with the processing
of APP by endogenous PS2. The ability of Alg3 to interfere with APP
processing and with apoptosis suggests that it is a general inhibitor
of PS2 function.
| |
DISCUSSION |
PS1 was recently shown to be necessary for -secretase activity;
however, its role in other aspects of APP processing is unclear (16).
We show that PS1 is also required for the coupling of APP processing to
signal transduction cascades and for an inhibitory regulation of
cleavage of APP at the -cleavage site. These roles of PS1 were
demonstrated by evaluating cells deficient in PS1, cells with normal
quantities of PS1, and cells overexpressing PS1. Our data show that the
effects due to loss of PS1 extend beyond reduction of the -secretase
cleavage. Cells lacking PS1 exhibit more cleavage of APP at or secretase sites under basal conditions, increased amounts of APP
C-terminal fragments, increased amounts of intracellular APPs, and
impaired coupling of APP processing to signal transduction cascades.
These changes in APP processing occur in both clonal, SV40 transformed
PS1 / cell lines and in primary cultures of PS1 / fibroblasts,
which shows that the changes in APP processing are not due to the
transformation process. Increasing PS1 expression produces the opposite
effect of loss of PS1 and reduces APP C-terminal fragments both in PS1
/ cells and in 293 HEK cells. Results from other studies have shown
that increasing PS1 expression increases APP secretion (28). These reciprocal changes in APP processing that are associated with increasing or decreasing PS1 levels points to a tight linkage between
PS1 expression and APP processing.
DeStrooper et al. (16) have shown that loss of PS1 leads to
reduced cleavage of APP at the -secretase site. Our results and
those of others confirm this observation (17, 29). A secretion is
reduced in PS1 / cells, which supports the observation that lack of
PS1 reduces cleavage of APP by -secretase. Despite reduced A
secretion, the total amount of APP cleavage products is also increased
in PS1 / cells, including increases in the levels of intracellular
APPs and APP C-terminal fragments. The increase in APP C-terminal
fragment and decrease in A secretion that we observed have also been
observed in neurons lacking PS1 and might be a general feature of cells
lacking PS1 (16, 17, 29). We observed that fibroblasts lacking PS1
produce more APPs, which is in agreement with the observations of
Naruse et al. (17). Interestingly, in the fibroblasts in our
study, the APPs is not secreted, whereas neurons lacking PS1 do secrete
the extra APPs that they produce (17). The increased levels of APP
cleavage products in PS1 / cells suggests that PS1 regulates
aspects of APP processing beyond the -secretase cleavage. An
important line of future studies will be to examine the regulation of
APPs in primary neurons.
A requirement for PS1 in the coupling of APP processing to signal
transduction pathways has not previously been shown. Cells lacking PS1
show deficits in the coupling of APP processing to signal transduction.
The PS1 / cells neither increase secretion of APP nor generate more
APP C-terminal fragments after stimulating PKC and extracellular
regulated kinase by treatment with PMA or EGF, respectively. The lack
of responsiveness of APP processing to PMA or EGF could result from a
reduced ability of the cell to cleave APP. However, transfecting APP
into PS1 / cells does increase APP secretion, which indicates that
PS1 / cells do have the capacity to increase APP cleavage and
secretion, despite not responding to PMA or EGF. The lack of
responsiveness of APP processing to PMA or EGF also cannot be explained
by poor receptor or signal transduction signaling, because signaling
from the EGF receptor through the extracellular regulated kinase
cascade to Elk gene transcription remains intact in the cells lacking
PS1. Given that the PS1 / cells are able to increase APP secretion after transfection of PS1 and that EGF signaling remains strong in the
PS1 knockout cells, the unresponsiveness of APP processing in PS1 /
appears to result from deficits in the coupling of APP processing to
signal transduction. Thus, PS1 appears to be required for normal
cleavage and secretion of APP, including coupling signal transduction cascades.
Cells expressing mutant PS1 associated with familial AD also are unable
to increase APP secretion after stimulation with PMA. However, this
phenotype appears to differ from the phenotype associated with loss of
PS1. Unlike PS1 / cells, cells expressing mutant PS1 have no
changes in levels of APP C-terminal fragments or basal secretion of
APP. The normal levels of APP cleavage products seen in cells
expressing mutant PS1 constructs suggest that the familial Alzheimer
mutations in PS1 do not cause a full loss of PS1 function. Other
studies suggest the same conclusion. For instance, mutant PS1
constructs are able to rescue the developmental lethality seen in PS1
knockout mice (although they are unable to correct cytoskeletal
defects) (30). PS1 mutant constructs are also able to partially rescue
the egg laying deficits seen in Sel12 mutants in the nematode (31, 32).
Thus, multiple lines of evidence indicate that the mutations in PS1
that cause familial AD do not cause a full loss of function phenotype.
The function of PS2 is also unknown. PS2 has been shown to bind APP,
and several studies suggest that PS2 regulates apoptosis (24) (26, 27).
A surprising outcome of our investigation is the observation that PS2
can compensate for the loss of PS1 in the PS1 / cells.
Overexpressing PS2 in PS1 / cells increases secretion of APP,
reduces basal production of APP C-terminal fragments, and restores
appropriate coupling of APP processing to PKC. These experiments
indicate that overexpressed PS2 has enough functional similarity to PS1
to restore the loss of APP processing in cells lacking PS1. One other
study has examined the effects of PS2 on APP processing in cells
expressing PS1; however, the effects of PS2 on these cells was not as
strong as the effects that we observed (33). That study (33) examined
the actions of the PS2 C-terminal fragment, Alg-3 but failed to observe
any effects of Alg3 on APP processing. In contrast, we observed that
transfecting Alg3 into PS1 / cells increases levels of APP
C-terminal fragments. This increase suggests that Alg3 is inhibiting
endogenous PS2 function, which is consistent with its effects on
apoptosis (25, 27, 34). The ability of PS2 and Alg3 to alter APP
processing in PS1 / cells suggests that PS2 is capable of
functioning like PS1 in regulating APP cleavage. The smaller effects of
PS2 and Alg3 on APP cleavage in cells expressing PS1 suggests that PS1 might mask some of the actions of PS2. As with many other redundant biological systems, PS2 might act as a backup for PS1.
Our data add to an increasing number of studies showing that PS1 and
PS2 regulate APP processing. There are several possible mechanisms that
are consistent with a broader role in APP processing. PS1 and PS2 might
directly regulate the activity of other secretases, such as and secretase. Another possibility is that presenilins do not
directly regulate the APP secretases but regulate the
compartmentalization of APP or its secretases. In this latter case,
loss of PS1 could lead to a different distribution of APP within the
microenvironment of the endoplasmic reticulum, which would alter its
availability to the proteins necessary for the catabolism of APP, APPs,
and APP C-terminal fragments. Our data also suggest that PS1 and PS2 are sensitive to the activity of PKC and ERK and that PS1 is necessary to allow APP processing to respond to changes in the activity of PKC
and ERK. The signal transduction coupling imparted by presenilins could
result either from control of the compartmentalization of APP, control
of the interaction of APP with secretases, or control of the activity
of secretases. The observation that PS1 and PS2 have a role in the
signal transduction coupling of APP processing adds to the increasing
number of reports indicating that presenilins interact with signal
transduction proteins quite extensively and are themselves substrates
of kinases (35-38). The potential physiological effects of such
interactions have generally been unclear, but the requirement of PS1
for coupling of APP processing to signal transduction suggests that
this interaction of PS1 with signal transduction enzymes might serve to
regulate APP processing.
The importance of PS1 and PS2 in regulating APP processing and the
ability of loss of PS1 or transfecting Alg3 to inhibit APP processing
suggest that inhibiting PS1 or PS2 in normal cells might reduce A
production, which could be beneficial because production of A is
generally thought to be necessary for the pathophysiology of AD. In
fact, it appears that the mutant D257A and D385A PS1 construct can
inhibit A production in CHO cells (18). Thus, inhibitors of PS1 or
PS2 might be potential therapeutic agents for AD. Whether PS1
inhibitors cause toxicity in adults is unknown, but loss of PS1 is
toxic to fetuses, as shown by the developmental lethality associated
with the PS1 / phenotype. Because PS1 inhibitors would be
administered to adults rather than to developing fetuses, the PS1
inhibition on adult physiology is not known. APP does not appear to be
necessary for neuronal function (39, 40), but PS1 does have actions
beyond APP processing. For instance, PS1 is required for normal
processing of NOTCH, glycosylation of the Trk B receptor is altered in
cells lacking PS1, and there is a possibility that apoptosis might also
be reduced (17, 41-45). The significance of such changes for
neurophysiology, though, is not known. These questions point out the
importance of understanding the impact of PS1 and PS2 on the adult brain.
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Dept. of
Pharmacology, Loyola University Medical Center, Bldg. 102, Rm. 3634, 2160 South 1st Ave., Maywood, IL 60153. Tel.: 708-216-6195; Fax: 708-216-6596; E-mail: bwolozi@wpo.it.luc.edu.
