From the Sanders-Brown Research Center on Aging and Department of
Anatomy and Neurobiology, University of
Kentucky, Lexington, Kentucky 40536
Mutations in the presenilin-1 (PS-1)
gene account for approximately 50% of the cases of autosomal dominant,
early onset, inherited forms of Alzheimer's disease (AD). PS-1 is an
integral membrane protein expressed in neurons and is localized
primarily in the endoplasmic reticulum (ER). PS-1 mutations may promote
neuronal degeneration by altering the processing of the
-amyloid
precursor protein (APP) and/or by engaging apoptotic pathways.
Alternative processing of APP in AD may increase production of
neurotoxic amyloid
-peptide (A
) and reduce production of the
neuroprotective
-secretase-derived form of APP (sAPP
). In
differentiated PC12 cells expressing an AD-linked PS-1 mutation
(L286V), sAPP
activated the transcription factor NF-
B and
prevented apoptosis induced by A
. Treatment of cells with
B decoy
DNA blocked the antiapoptotic action of sAPP
, demonstrating the
requirement for NF-
B activation in the cytoprotective action of
sAPP
. Cells expressing mutant PS-1 exhibited an aberrant pattern of
NF-
B activity following exposure to A
, which was characterized by
enhanced early activation of NF-
B followed by a prolonged depression
of activity. Blockade of NF-
B activity in cells expressing mutant
PS-1 by
B decoy DNA was associated with enhanced A
-induced
increases of [Ca2+]i and mitochondrial
dysfunction. Treatment of cells with sAPP
stabilized
[Ca2+]i and mitochondrial function and suppressed
oxidative stress by a mechanism involving activation of NF-
B.
Blockade of ER calcium release prevented (and stimulation of ER calcium release by thapsigargin induced) apoptosis in cells expressing mutant
PS-1, suggesting a pivotal role for ER calcium release in the
proapoptotic action of mutant PS-1. Finally, a role for NF-
B in
preventing apoptosis induced by ER calcium release was demonstrated by
data showing that sAPP
prevents thapsigargin-induced apoptosis, an
effect blocked by
B decoy DNA. We conclude that sAPP
stabilizes
cellular calcium homeostasis and protects neural cells against the
proapoptotic action of mutant PS-1 by a mechanism involving activation
of NF-
B. The data further suggest that PS-1 mutations result in
aberrant NF-
B regulation that may render neurons vulnerable to
apoptosis.
 |
INTRODUCTION |
Alzheimer's disease
(AD)1 is characterized by the
accumulation of amyloid
-peptide (A
) and death of neurons in
brain regions involved in learning and memory processes (1). Neuronal
apoptosis (2, 3) is implicated in AD based on studies of postmortem brain tissues (4, 5) and cell culture studies showing that A
can
induce apoptosis (6, 7). Some cases of AD are inherited in an autosomal
dominant manner and are characterized by early age of onset;
approximately half of these cases are caused by mutations in the
presenilin-1 (PS-1) gene located on chromosome 14 (8-10). PS-1 encodes
an integral membrane protein with six or eight membrane-spanning
domains (11-13) and is localized in the endoplasmic reticulum (ER;
Refs. 14-17). PS-1 is expressed in neurons throughout the brain
(18-21) and is present in both degenerating and nondegenerating
neurons in AD brain (22, 23). Consequences of PS-1 and PS-2 mutation
expression in cultured cells include increased production of a long
form of A
(A
-(1-42); Refs. 24 and 25), decreased choline
acetyltransferase activity (26), and increased vulnerability of cells
to apoptosis (27-30). The mechanism(s) whereby PS mutations sensitize
neurons to apoptosis is unknown.
Links between aberrant proteolytic processing of APP and neuronal
degeneration in AD are supported by considerable data (for a review,
see Ref. 31). APP mutations, which account for some cases of inherited
AD, may simultaneously increase production of neurotoxic forms of A
and decrease production of neuroprotective secreted forms of APP
(sAPP
). A
can induce neuronal apoptosis and can increase neuronal
vulnerability to excitotoxicity by a mechanism involving induction of
oxidative stress (32-36) and disruption of calcium homeostasis
(35-39). On the other hand, sAPP
stabilizes neuronal calcium
homeostasis and protects neurons against excitotoxic, metabolic, and
oxidative insults including exposure to A
(40, 41).
It was recently reported that sAPP
induces activation of the
transcription factor NF-
B in cultured hippocampal neurons (42). NF-
B exists in the cytosol as an inducible three-subunit complex consisting of the transcription factor dimer (p50 and p65) and an
associated inhibitory subunit called I-
B (43). NF-
B activation occurs when I-
B is induced to dissociate from the complex, a process
that may involve phosphorylation, proteolysis, and/or oxidative damage
to I-
B. Recent findings suggest that NF-
B plays an antiapoptotic
role in nonneuronal cells (44, 45) and in neurons (46-48). Agents that
activate NF-
B (e.g. tumor necrosis factor and ceramide)
can prevent cell death induced by excitotoxic, metabolic, and oxidative
insults including exposure to A
(49, 50), whereas
B decoy DNA
(which blocks NF-
B activity) enhances cell death in several
paradigms (47, 48). NF-
B activity may be altered in vulnerable brain
regions in AD patients, such that its activity is increased in neurons
associated with plaque amyloid (51, 52). We now report that sAPP
counteracts the proapoptotic actions of mutant PS-1 by activating
NF-
B and stabilizing calcium homeostasis. NF-
B appears to
interrupt the apoptotic program at an early stage prior to oxyradical
production and mitochondrial dysfunction. An abnormal sustained
suppression of NF-
B activity following exposure of cells to A
occurred in cells overexpressing mutant (but not wild-type) PS-1,
suggesting a role for this transcription factor in the pathogenic
mechanism of PS-1 mutations.
 |
MATERIALS AND METHODS |
PC12 Cell Lines and Experimental Treatments--
Rat
pheochromocytoma (PC12) cell lines stably expressing human wild-type
PS-1 and mutant PS-1 (L286V) were established using the
"Tet-off" expression system (Promega), where the expression of the transgene is under the control of a tetracycline-sensitive transactivator, as described in detail in our previous studies (28).
Cells were maintained at 37 °C (5% CO2 atmosphere) in RPMI medium supplemented with 10% heat-inactivated horse serum and 5%
heat-inactivated fetal bovine serum. For differentiation, cells were
maintained in the presence of 50 ng/ml nerve growth factor for 14 days
in RPMI containing 2% bovine serum albumin in the continued presence
of 2 µg/ml tetracycline; the tetracycline was then removed for
48 h to induce PS-1 expression. Preliminary Western blot analyses
demonstrated responsiveness of PS-1 expression to tetracycline
withdrawal and that lines expressing wild-type and mutant PS-1
overexpressed the proteins at comparable, moderately high, levels (28);
two lines overexpressing wild-type PS-1 (PS-1C3 and PS-1C7) and two
lines overexpressing the L286V PS-1 mutation (PS1L286VC1 and
PS1L286VC9) at similar levels were used in the present study.
Synthetic A
-(1-42) was synthesized at the University of Kentucky
Macromolecular Analysis Facility and purified by high pressure liquid
chromatography, and it was stored lyophilized; a 1 mM stock solution was prepared in sterile deionized water approximately 16 h prior to use. Immediately prior to experimental treatment, the
culture medium was switched to Locke's solution, which contained 154 mM NaCl; 5.6 mM KCl, 2.3 mM
CaCl2, 1.0 mM MgCl2, 3.6 mM NaHCO3, 10 mM glucose, 5 mM Hepes buffer (pH 7.2). Secreted APP
(sAPP
695) was
purified from the culture supernatant of human embryonic kidney 293 cells transfected with the corresponding cDNA constructs as described previously (40). Purity was confirmed by Western blot analysis and silver staining after SDS-polyacrylamide gel
electrophoresis. Double-stranded
B decoy DNA was prepared by
annealing complementary single strands (2 mM each) with the
sequences 5'-GAGGGGACTTTCCCT-3' and 5'-AGGGAAAGTCCCCTC-3'. Nifedipine,
sodium dantrolene, and thapsigargin were purchased from Sigma and
prepared as 500 × stocks in ethanol.
Quantification of Apoptosis and Mitochondrial
Function--
Methods for analysis of apoptosis are detailed in our
previous studies (28, 36). Briefly, cells were stained with the fluorescent DNA-binding dyes Hoechst 33342 or propidium iodide, and
cells were visualized under epifluorescence illumination. Cells with
condensed and fragmented (apoptotic) nuclei were counted in four
random × 40 fields per culture; counts were made without knowledge of cell line or treatment history. Images of propidium iodide-stained cells were acquired with a confocal laser scanning microscope (Molecular Dynamics; 488-nm excitation and 510-nm barrier filter) using a × 60 oil immersion objective. Two methods were employed to assess mitochondrial function. Mitochondrial transmembrane potential was assessed using the dye rhodamine 123 (Molecular Probes,
Inc.) as described previously (53). Cultures were incubated for 30 min
in RPMI 1640, containing 5 µM rhodamine 123, and were then washed with Locke's solution. Cellular fluorescence was imaged using a laser scanning confocal microscope with excitation at 488 nm
and emission at 510 nm. Levels of cellular MTT reduction, a measure of
mitochondrial energy charge/redox state (54), were quantified as
described previously (36). Briefly, MTT solution (5 mg/ml
phosphate-buffered saline) was added to cultures (1:10, MTT
solution:culture medium, v/v) and allowed to incubate for 3 h. The
cells were washed three times with Locke's solution and solubilized in
dimethyl sulfoxide, and the absorbance (592 nm) in each well was
quantified using a plate reader.
Measurements of [Ca2+]i and Cellular
Peroxide Levels--
Fluorescence ratio imaging of the calcium
indicator dye fura-2 was performed as described previously (37, 38).
Briefly, following exposure to A
, cells were loaded with fura-2
(30-min incubation in the presence of 10 µM fura-2), and
maintained in the presence of A
and other treatments during imaging.
Fluorescence was imaged using a Zeiss AttoFluor system with a × 40 oil objective; the average [Ca2+]i in
individual neuronal cell bodies was determined from the ratio of the
fluorescence emissions obtained using two different excitation
wavelengths (334 and 380 nm). The system was calibrated using solutions
containing either no Ca2+ or a saturating level of
Ca2+ (1 mM) using the formula
[Ca2+]i = Kd[(R
Rmin)/(Rmax
R)](Fo/Fs). Relative
levels of cellular peroxides were quantified in individual PC12 cells
by confocal laser scanning microscope analysis using the dye
2',7'-dichlorofluorescin diacetate (Molecular Probes) as described
previously (33). Briefly, cells were incubated for 50 min in the
presence of 50 µM dye and were then washed three times (2 ml/wash) in Locke's solution. Images of cellular fluorescence were
acquired using a confocal laser scanning microscope (Molecular Dynamics) with excitation at 488 nm and emission at 510 nm. The intensity of the laser beam and the sensitivity of the photodetector were held constant to allow quantitative comparisons of relative fluorescence intensity of cells between treatment groups. Cells were
located under visible light and scanned only once with the laser to
avoid artifacts associated with photo-oxidation. Fluorescence intensities in cell bodies were quantified using Molecular Dynamics "Imagespace" software.
Electrophoretic Mobility Gel Shift Assay--
These methods were
similar to those described previously (46, 48). Briefly, cell extracts
containing DNA-binding proteins were prepared, and gel shift assays
were performed using a commercially available assay kit (Promega).
Double-stranded, 32P-labeled DNA (
B consensus sequence
5'-AGT TGA GGG GAC TTT CCC AGG C-3'; 100,000 cpm) was added to a
reaction mixture containing 5 µl nuclease-free water, 2 µl of gel
shift binding 5× buffer (20% glycerol, 5 mM
MgCl2, 2.5 mM EDTA, 2.5 mM
dithiothreitol, 250 mM NaCl, 0.25 mg/ml
poly(DL-dC)-poly(DL-dC), and 50 mM
Tris-HCl, pH 7.5) and 2 µl (5 µg) of cell extract. The reaction was
allowed to proceed for 20 min at room temperature, and then 1 µl of
gel loading 10× buffer (40% glycerol, 0.2% bromphenol blue, 250 mM Tris-HCl, pH 7.5) was added. Samples were separated by
electrophoresis through a nondenaturing 4% acrylamide gel, and the gel
was dried and exposed to x-ray film. To demonstrate the specificity of
the inducible NF-
B complexes in PC12 cells overexpressing PS-1
mutation detected in gel shift reactions, we performed five reactions: a positive control (where the 32P-labeled specific NF-
B
consensus oligonucleotide was included in the binding reaction), a
negative control (where no DNA-binding protein extract was added in the
binding reaction), a specific competition assay (where an excess of
unlabeled specific competitor NF-
B oligonucleotide probe was
included in the binding reaction), a mutant
B competition assay
(where an excess of unlabeled mutant NF-
B oligonucleotide 5'-AGT TGA
GGC GAC TTT CCC AGG C-3' (Santa Cruz, CA) was included in the binding
reaction), and a nonspecific competition assay (where excess unlabeled
noncompetitor AP2 consensus oligonucleotide 5'-GAT CGA ACT GAC CGC CCG
CGG CCC GT-3' was included in the binding reaction). To further
determine the specificity of the gel shift reaction and to examine the
possible subunit composition of the inducible NF-
B complexes, we
performed supershift experiments using antibody raised against p50 or
p65 proteins purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
The anti-p50 antibody was rabbit IgG raised against an epitope
corresponding to amino acids 350-363 within the nuclear localization
signal region of human p50. The anti-p65 antibody was rabbit IgG raised against the N-terminal amino acids 3-19 of the human p65. As a control
for p50 and p65 antibodies, extracts of DNA-binding proteins were also
incubated in the presence of preimmune rabbit serum. Supershift
experiments were performed by adding 1.0 µl of the antibody per 10 µl of reaction volume prior to the addition of 32P-labeled oligonucleotide probe, and the mixtures were
incubated overnight at 4 °C. Relative levels of DNA-protein hybrids
were quantified by densitometric analysis of scanned gel images using NIH Image 1.47 software.
 |
RESULTS |
sAPP
Protects PC12 Cells Expressing Mutant PS-1 against
Apoptosis Induced by A
--
We examined the effects of mutant PS-1
and sAPP
on vulnerability of PC12 cells to apoptosis induced by A
(A
-(1-42)) using two cell lines overexpressing wild-type human PS-1
(PS-1C3 and PS-1C7), two lines expressing the L286V PS-1 mutation
(PS-1L286VC1 and PS-1L286VC9), a vector-transfected control line, and
the untransfected parent cell line. Preliminary Western blot analysis
showed that levels of overexpression of wild-type and mutant PS-1
48 h following withdrawal of tetracycline were similar in the
selected cell lines under the conditions employed (data not shown;
cf. Ref. 28). Basal levels of apoptosis in the various cell
lines ranged from 1 to 4% (Fig. 1). In
previous concentration-effect studies, we found that an A
-(1-42)
concentration of 50 µM reliably induced a moderate level
of apoptosis in untransfected PC12 cells during 24-48-h exposure
periods (28, 36). Exposure to 50 µM A
for 48 h
induced apoptosis in 30-40% of cells in control PC12 cell lines and
lines expressing wild-type PS-1; apoptosis was significantly enhanced
(to 60-70%) in lines expressing mutant PS-1 (Figs. 1 and
2). Mutations in APP responsible for some
inherited forms of AD may promote neuronal degeneration by increasing
levels of neurotoxic A
and/or by decreasing production of sAPP
,
which has been shown to protect neurons against excitotoxic and
oxidative insults (for a review, see Ref. 31). When cell lines were
pretreated for 24 h with 10 nM sAPP
, there was a
highly significant suppression of A
-induced apoptosis in both
control lines and lines expressing mutant PS-1 (Fig. 1).

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Fig. 1.
Secreted APP protects PC12 cells against
the proapoptotic action of mutant PS-1: evidence for the involvement of
NF- B activation and stabilization of calcium homeostasis. The
indicated lines of PC12 cells were pretreated for 24 h with
sAPP (10 nM) or B decoy DNA (25 µM), or
for 1 h with dantrolene (DTL; 10 µM) or
nifedipine (Nif; 1 µM) and were then exposed
to A (50 µM) for 48 h, and apoptosis was
quantified (see "Materials and Methods"). PC12 cell lines analyzed
included untransfected cells (A), vector-transfected cells
(B), two different lines overexpressing wild-type PS-1
(C and D), and two different lines overexpressing
mutant PS-1 (E and F). Values are the mean and
S.E. of determinations made in 4-6 separate cultures. *,
p < 0.01 compared with control and sAPP values. **,
p < 0.01 compared with the values for cultures exposed
to A , A plus decoy DNA, and sAPP plus A plus decoy DNA. For
panels A-D the value for cells exposed to A plus decoy
DNA was significantly greater than the value for cultures exposed to
A alone (p < 0.05). Values for PS-1L286VC1 and
PS-1L286VC9 lines exposed to A , A plus decoy DNA, and A plus
sAPP plus decoy DNA were significantly greater than corresponding
values in each of the other cell lines (p < 0.05)
(ANOVA with Scheffe's post hoc tests).
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Fig. 2.
Confocal laser scanning microscope images of
PC12 cells (expressing mutant PS-1) stained with the fluorescent
DNA-binding dye propidium iodide. The DNA in cells in control
cultures and cultures pretreated with sAPP and then exposed to A
for 48 h is diffusely distributed throughout the nucleus
(arrowhead), whereas the DNA is condensed and fragmented
48 h following exposure to 50 µM A alone and in
cultures pretreated with decoy DNA plus sAPP and then exposed to
A (arrows).
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Involvement of NF-
B in the Antiapoptotic Action of
sAPP
--
A variety of intercellular signals that can prevent cell
death induce activation of the transcription factor NF-
B in neural cells including tumor necrosis factor-
(TNF
) (46), neural growth
factor (47), and sAPP
(42). NF-
B is also activated in cells
exposed to a variety of insults, including A
, that induce oxidative
stress and increase [Ca2+]i (32, 52), which may
represent a stress response designed to protect the cells. In order to
determine whether NF-
B activation mediated the antiapoptotic action
of sAPP
in PC12 cells, we employed
B decoy DNA to block
activation of NF-
B (48, 55). Whereas
B decoy DNA alone had no
effect on basal levels of apoptosis (data not shown), it significantly
enhanced A
-induced apoptosis and completely abolished the
antiapoptotic effect of sAPP
(Fig. 1). We next performed gel shift
analysis to measure levels of NF-
B activity following exposure to
A
and sAPP
in the different PC12 cell lines. In agreement with a
previous study (56), we observed two shifted bands representing
B-binding proteins in unstimulated PC12 cells (Fig.
3A). The intensity of the
bands was also markedly reduced in cultures treated with 25 µM
B decoy DNA (Fig. 3A). Gel shift
analysis showed that both A
and sAPP
induced NF-
B activation
and that
B decoy DNA prevented activation of NF-
B by each agent
(Figs. 3, B-D, and 4). The
two shifted bands were specific
B-binding proteins, since they were eliminated by incubation with excess cold specific
B competitor DNA
but not by cold mutant
B or nonspecific (AP2) competitor oligonucleotide (Fig. 3E). Supershift analysis demonstrated
that the higher molecular weight band of NF-
B was supershifted by the p65 antibody, while the lower molecular weight band of NF-
B was
supershifted by the p50 antibody, indicating that the higher molecular weight band was p65, while the lower molecular weight band was p50 (Fig. 3E).

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Fig. 3.
Effects of mutant PS-1 and sAPP on NF- B
activity following exposure of PC12 cells to A . A, PC12
cells were exposed for 24 h to vehicle (Control) or 25 µM B decoy DNA. Cell extracts were subjected to gel
shift analysis. Specificity of the protein-DNA interactions
representing the shifted bands was established in samples in which
excess cold competitor DNA (lane 4) or nonspecific
(scrambled) DNA (lane 2) was included in the assay and by not adding cell extract to the
reaction (lane 3). The arrowheads point to the
shifted bands corresponding to NF- B subunits, and the
arrow points to the free DNA. B, PC12 cell lines
(untransfected cells (top), vector-transfected cells
(middle), and cells overexpressing wild-type PS-1
(bottom)) were exposed to vehicle for 4 h or to 50 µM A for the indicated time periods (in the absence or
presence of 25 µM B decoy DNA), and cell extracts were
subjected to gel shift analysis. C, autoradiograms from a
gel shift analysis of cell extracts from PC12 cells expressing mutant
PS-1 that had been exposed to vehicle for 4 h or to 50 µM A for the indicated time periods (in the absence or
presence of 25 µM B decoy DNA). D,
autoradiograms from a gel shift analysis of cell extracts from PC12
cells expressing mutant PS-1 that had been exposed to vehicle for
4 h (Control) or to the indicated treatments for the
indicated time periods. Concentrations were as follows: A , 50 µM; sAPP , 10 nM; B decoy DNA, 25 µM. E, PC12 cells overexpressing PS-1 L286V
were stimulated for 4 h with 50 µM of A , and
DNA-binding proteins were extracted and subjected to EMSA and
supershift EMSA. Positive Control, 32P-labeled
specific NF- B consensus oligonucleotide (5'-AGT TGA GGG GAC TTT CCC
AGG C-3'), was included in the binding reaction); Negative
Control, no DNA-binding protein extract was present in the
reaction; Specific Competitor, excess unlabeled specific
competitor NF- B consensus oligonucleotide was included in the
reaction; Mutant Competitor, an excess of unlabeled mutant
NF- B oligonucleotide (5'-AGT TGA GGC GAC TTT CCC AGG C-3') was
included in the reaction; Nonspecific Competitor, excess
unlabeled noncompetitor (AP2 consensus oligonucleotide 5'-GAT CGA ACT
GAC CGC CCG CGG CCC GT-3') was included in the reaction. Supershift
experiments were performed using antibodies against p50 and p65
proteins (see "Materials and Methods"). Note the higher molecular
weight band of NF- B was supershifted by the p65 antibody, while the
lower molecular weight band of NF- B was supershifted by the p50
antibody, indicating that the higher molecular weight band was p65,
while the lower molecular weight band was p50. As a control for p50 and
p65 antibodies, extracts were incubated in the presence of preimmune
rabbit serum.
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Fig. 4.
Densitometric analysis of B DNA-binding
activities corresponding to the upper shifted band (A and
B) and the lower shifted band (C and
D) (see Fig. 3). The time courses of changes in levels
of each active B-binding protein following exposure to 50 µM A in control cultures (A and
C) and in cultures pretreated for 24 h with 10 nM sAPP (B and D) are shown.
Values are expressed as a percentage of the level in untreated control
cultures and represent the mean and S.E. of determination made in three
or four separate experiments. The values for PC12 cells expressing
mutant PS-1 were significantly different from the values for each of
the other cell lines as follows. A, 4 h
(p < 0.01), 8 h (p < 0.05),
24 h (p < 0.01), and 48 h (p < 0.05); B, 4 h (p < 0.01);
C, 4 h (p < 0.001), 24 h
(p < 0.01), 48 h (p < 0.01);
D, 4 h, p < 0.01, 24 h
(p < 0.05) (ANOVA with Scheffe's post
hoc tests). Overexpression of either wild-type or mutant PS-1 did
not affect basal NF- B activity under normal culture
conditions.
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We next examined the pattern of NF-
B activation in response to A
in control PC12 cell lines and lines expressing mutant PS-1. Control
cell lines and lines expressing mutant PS-1 were exposed to A
for
different time periods, and levels of NF-
B activity were quantified
by gel shift analysis. Marked, but transient, increases in NF-
B
activity occurred within 4 h of exposure to A
in each cell
line; levels of DNA binding activity of each of two different bands
were increased in cells exposed to A
(Figs. 3 and 4). The magnitude
of the early NF-
B induction was greater in lines expressing mutant
PS-1 than in control lines, a result consistent with increased levels
of oxidative stress and [Ca2+]i in cells
expressing mutant PS-1 (see below). However, levels of NF-
B activity
decreased progressively 8, 24, 48, and 72 h following exposure to
A
in all cell lines. Notably, levels of NF-
B activity 24-48 h
following exposure to A
were significantly lower in cells expressing
mutant PS-1 compared with control cell lines (Figs. 3 and 4). There was
a correlation between a very low level of NF-
B activity beginning at
8 h after A
treatment in the lower shifted band and 24 h
post-A
in the upper shifted band and increased apoptosis in cells
expressing mutant PS-1 (Figs. 1 and 4). Treatment of cells expressing
mutant PS-1 with sAPP
prevented the prolonged suppression of NF-
B
activity that occurred 24-48 h following exposure to A
in the
absence of sAPP
, and, in fact, levels of NF-
B activity remained
higher than in unstimulated control cultures (Figs. 3D and
4). Collectively, these findings suggest a role for sustained increases
in NF-
B activity in the cytoprotective action of sAPP
.
Cellular Calcium Homeostasis Is Disrupted by Mutant PS-1 and
Restored by sAPP
--
Because elevations of
[Ca2+]i may mediate A
-induced apoptosis (34,
37, 38) and because sAPP
has been shown to stabilize
[Ca2+]i in neurons exposed to various insults
(40, 41), we examined the effects of agents that suppress calcium
release from ER (dantrolene) or influx through plasma membrane
voltage-dependent channels (nifedipine) on apoptosis
induced by A
. Dantrolene and nifedipine each prevented apoptosis in
control PC12 cell lines and in lines expressing mutant PS-1 (Fig. 1),
consistent with roles for calcium release from ER and influx through
voltage-dependent membrane channels in the apoptotic
process.
B decoy DNA did not alter the abilities of dantrolene and
nifedipine to prevent A
-induced apoptosis (Fig. 1).
We next measured [Ca2+]i by fluorescence ratio
imaging of the calcium indicator dye fura-2 in PC12 cell lines exposed to A
(Fig. 5). Four hours following
exposure of control and wild-type PS-1-overexpressing cell lines to
A
, the [Ca2+]i was increased to approximately
200 nM, compared with 90-100 nM in cells not
exposed to A
(Fig. 5A). The [Ca2+]i
increase in response to A
was markedly enhanced in cells expressing
mutant PS-1, with levels reaching 400 nM within 4 h of
exposure (Fig. 5A). Pretreatment of cells with sAPP
resulted in a significant attenuation of A
-induced
[Ca2+]i increases in each cell line, with the
magnitude of the effect being greatest in lines expressing mutant PS-1
(Fig. 5). When cells were cotreated with
B decoy DNA and sAPP
, no attenuation of A
-induced elevation of [Ca2+]i
was observed (Fig. 5A), suggesting that NF-
B mediated the
[Ca2+]i-stabilizing action of sAPP
.
Co-treatment of cells of each line with
B decoy DNA plus A
resulted in a significantly greater [Ca2+]i
compared with cells exposed to A
alone (Fig. 5A), suggesting that NF-
B activation in response to A
played a role in
stabilizing cellular calcium homeostasis. Exposure of the different cell lines (untransfected, vector-transfected, wild-type PS-1, and
mutant PS-1) to
B decoy DNA alone caused no change in
[Ca2+]i during exposures of up to 12 h (Fig.
5B). However, 24 h following exposure to
B decoy
DNA, there was a significant 2-fold increase of
[Ca2+]i in cells expressing mutant PS-1 but not
in control cell lines and lines overexpressing wild-type PS-1 (Fig.
5B). The latter result suggests that basal levels of NF-
B
activity play a role in counteracting the adverse effect of mutant PS-1 on cellular calcium homeostasis.

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Fig. 5.
Secreted APP suppresses the elevation of
[Ca2+]i induced by A in PC12 cells expressing
mutant PS-1. A, the indicated cell lines were exposed to
vehicle (Control) or 50 µM A for 4 h;
additional cultures were pretreated with 10 nM sAPP
(alone or in combination with 25 µM B decoy DNA) prior
to exposure to A . The [Ca2+]i was then
quantified by imaging of the calcium indicator dye fura-2. Values are
the mean and S.E. of determinations made in three or four cultures
(20-30 cells analyzed per culture). *, p < 0.01 compared with corresponding values for cells not exposed to A . **,
p < 0.01 compared with values for corresponding cell
lines exposed to A alone. B, the indicated cell lines
were exposed to 25 µM B decoy DNA, and the
[Ca2+]i was quantified at the indicated time
points thereafter. Values are the mean and S.E. of determinations made
in three or four cultures (20-30 cells analyzed per culture). *,
p < 0.01 compared with each of the other three values at
that time point. Similar results were obtained in a separate experiment
involving lines PS-1C7 and PS-1L286VC9.
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Recent data from studies of nonneuronal cells suggest that ER calcium
regulation plays a role in apoptosis in several paradigms (57, 58).
Because PS-1 is located in the ER, and because dantrolene protected
PC12 cells against the proapoptotic action of mutant PS-1 (Fig. 1),
we determined whether sAPP
would protect cells against apoptosis
induced by thapsigargin, an agent known to induce apoptosis by
selectively inhibiting the ER Ca2+-ATPase (59).
Pretreatment with sAPP
resulted in a significant reduction in
apoptosis induced by thapsigargin in each control cell line and in
lines expressing mutant PS-1 (Fig. 6).
The protective effect of sAPP
against thapsigargin-induced apoptosis
was abolished in cultures co-treated with
B decoy DNA (data not
shown) indicating a necessary role for NF-
B activation in
suppression of apoptosis induced by ER calcium release.

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Fig. 6.
Secreted APP suppresses apoptosis induced
by thapsigargin in PC12 cells expressing mutant PS-1. The
indicated cell lines were exposed to vehicle (Control) or 5 µM thapsigargin (Thaps) for 24 h; some
cultures were pretreated for 24 h with 10 nM sAPP
prior to exposure to thapsigargin. Cells were then stained with Hoechst
dye, and numbers of apoptotic cells in each culture were quantified.
Values are the mean and S.E. of determinations made in three or four
cultures. *, p < 0.01 compared with corresponding
values for untransfected (untransf), vector, PS-1C3, and
PS-1C7 lines. **, p < 0.01 compared with values for
corresponding cell lines exposed to thapsigargin alone.
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Enhanced Mitochondrial Dysfunction and Oxidative Stress in PC12
Cells Expressing Mutant PS-1: Protection by sAPP
--
Mitochondrial
alterations, including a decrease in mitochondrial transmembrane
potential and energy charge/redox state, have been identified as being
central to the effector phase of apoptosis (3) and may contribute to
the neurotoxic action of A
(36, 54). Exposure of PC12 cells to A
for 4 h resulted in decreases in levels of mitochondrial membrane
potential, as quantified using the fluorescent probe rhodamine 123 (Fig. 7A). The decrease in rhodamine 123 fluorescence was significantly greater in cells expressing mutant PS-1 compared with control cell lines and lines overexpressing wild-type PS-1 (Fig. 7A). Pretreatment of
cultures with sAPP
prior to exposure to A
resulted in a
significant attenuation of the decrease in transmembrane potential,
which was particularly pronounced in cell lines expressing mutant PS-1
(Fig. 7, A and C).
B decoy DNA suppressed the
ability of sAPP
to prevent A
-induced decrease in rhodamine 123 fluorescence (Fig. 7A) and enhanced the decrease in
rhodamine 123 fluorescence induced by A
. Exposure of the different
cell lines to
B decoy DNA alone caused no change in rhodamine 123 fluorescence during exposures of up to 12 h (Fig. 7B).
However, 24 h following exposure to
B decoy DNA, there was a
significant decrease in mitochondrial transmembrane potential in cells
expressing mutant PS-1 but not in control cell lines and lines
overexpressing wild-type PS-1 (Fig. 7B). The latter result
suggests that basal levels of NF-
B activity play a role in
counteracting an adverse effect of mutant PS-1 on mitochondrial function. In parallel experiments, we quantified levels of MTT reduction, a measure of mitochondrial energy charge/redox state (54),
in the different cell lines following exposure to A
in the presence
or absence of sAPP
and/or
B decoy DNA. Exposure of PC12 cells to
A
for 4 h resulted in a decrease in the level of MTT reduction
that was significantly greater in cells expressing mutant PS-1 compared
with control cell lines; pretreatment with sAPP
resulted in a
significant attenuation of A
-induced decrease in MTT reduction, and
this action of sAPP
was largely blocked by
B decoy DNA (data not
shown).

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Fig. 7.
Secreted APP protects against A -induced
decrease in mitochondrial transmembrane potential by a mechanism
involving activation of NF- B. A, the indicated lines of
PC12 cells were pretreated for 24 h with sAPP (10 nM) or B decoy DNA (25 µM) in the absence
or presence of sAPP and were then exposed to A (50 µM) for 4 h. Levels of rhodamine 123 fluorescence
were then quantified (see "Materials and Methods"). Values are the
mean and S.E. of determinations made in four separate cultures (25-35
cells analyzed per culture). *, p < 0.01 compared with
the control value. **, p < 0.01 compared with the
corresponding values for untransfected, vector-transfected, PS-1C3, and
PS-1C7 cell lines. (ANOVA with Scheffe's post-hoc tests).
B, the indicated cell lines were exposed to 25 µM B decoy DNA, and levels of rhodamine 123 fluorescence were quantified at the indicated time points thereafter.
Values are the mean and S.E. of determinations made in three or four
cultures (25-35 cells analyzed per culture). *, p < 0.01 compared with each of the other three values at that time point
(ANOVA with Scheffe's post hoc tests). Similar results were
obtained in a separate experiment involving lines PS-1C7 and
PS-1L286VC9. C, confocal laser scanning microscope images of
rhodamine 123 fluorescence in PC12 cells expressing mutant PS-1.
Cultures were pretreated for 24 h with 0.2% saline or 10 nM sAPP and were then exposed to either 0.5% water
(control and sAPP ) or 50 µM A -(1-42) for 4 h.
Relative levels of rhodamine 123 fluorescence are indicated by the
scale bar (lower right). Note that A caused a
marked decrease in levels of rhodamine 123 fluorescence, which was
attenuated in cells pretreated with sAPP .
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Previous studies showed that A
induces oxidative stress in cultured
neurons (32-36) and that this action of A
is enhanced in PC12 cells
expressing mutant PS-1 (28). In order to determine whether sAPP
modifies levels of oxidative stress and if NF-
B is involved, we
measured relative levels of cellular peroxides using the fluorescent
probe DCF (33). Exposure of PC12 cells to A
for 4 h resulted in
an increase in DCF fluorescence that was significantly greater in cells
expressing mutant PS-1 compared with control cell lines and lines
overexpressing wild-type PS-1 (Fig.
8A). The A
-induced increase
in DCF fluorescence was blocked in cells pretreated with sAPP
(Fig.
8). No suppression of A
-induced DCF fluorescence occurred in
cultures cotreated with
B decoy DNA and sAPP
(data not
shown).

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Fig. 8.
Secreted APP attenuates A -induced
peroxide accumulation in PC12 cells expressing mutant PS-1.
A, levels of DCF fluorescence, a measure of cellular
peroxide levels, were quantified in PC12 cells of the indicated lines.
Cultures were exposed for 4 h to either vehicle (0.5% water) or
50 µM A alone or in the presence of 10 nM
sAPP (sAPP was added to the cultures 24 h prior to exposure
to A ). Values are the mean and S.E. of determinations made in three
or four separate cultures (20-30 cells assessed per culture). *,
p < 0.01 compared with value for corresponding cell
line exposed to vehicle and to sAPP plus A . **, p < 0.001 compared with the value for the corresponding cell line exposed
to vehicle and p < 0.01 compared with values for
control cell lines (untransfected and vector-transfected lines and
lines overexpressing wild-type PS-1) exposed to A . ***,
p < 0.01 compared with corresponding cell lines
exposed to A alone (ANOVA with Scheffe's post hoc
tests). B, confocal laser scanning microscope images of DCF
fluorescence in PC12 cells expressing mutant PS-1. Cultures were
pretreated for 24 h with 0.2% saline or 10 nM sAPP
and were then exposed to either 0.5% water (control and sAPP ) or 50 µM A -(1-42) for 4 h. Relative levels of DCF
fluorescence are indicated by the scale bar (lower
right). Note that A caused a marked increase in levels of DCF
fluorescence, which was much less pronounced in cells pretreated with
sAPP .
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DISCUSSION |
The present findings demonstrate that sAPP
can protect PC12
cells against the proapoptotic action of mutant PS-1 by a mechanism involving activation of NF-
B, stabilization of calcium homeostasis and mitochondrial function, and suppression of oxidative stress. Recent
studies of tumor cells (44, 45, 60) and cultured embryonic rat
hippocampal neurons (48) have shown that activation of NF-
B (in
response to TNF
) can prevent apoptosis induced by several different
insults including exposure to oxidative insults. We found that sAPP
induces NF-
B activation in PC12 cells and protects them from being
killed by A
. The cytoprotective effect of sAPP
was largely
prevented by co-treatment with
B decoy DNA, a result that provides
evidence for a cause-effect relationship between activation of NF-
B
and prevention of apoptosis. The enhancement of A
-induced apoptosis
in PC12 cells overexpressing mutant human PS-1 was abrogated in
cultures pretreated with sAPP
, an effect blocked by
B-decoy DNA.
Previous studies have shown that the mechanism whereby A
damages and
kills neurons involves induction of oxidative stress, disruption of
calcium homeostasis, and mitochondrial dysfunction (for a review, see
Ref. 31). We found that A
caused increases of
[Ca2+]i and peroxide levels and induced
depolarization of the mitochondrial membrane and that these actions of
A
were each exacerbated in cells overexpressing mutant but not
wild-type PS-1. PC12 cells pretreated with sAPP
, including lines
expressing mutant PS-1, were able to maintain
[Ca2+]i, peroxide levels, and mitochondrial
transmembrane potential at nearly normal levels following exposure to
A
. The ability of
B decoy DNA to counteract the stabilizing
effect of sAPP
on [Ca2+]i, peroxide levels,
and mitochondrial transmembrane potential indicates a central role for
NF-
B in the antiapoptotic mechanism of action of sAPP
.
Gel shift analyses of NF-
B activation following exposure of PC12
cells to A
revealed differences in the pattern of NF-
B activity
in cells overexpressing mutant PS-1 compared with cells overexpressing
wild-type PS-1 and control cell lines. A striking consequence of mutant
PS-1 expression was a sustained suppression of NF-
B activity, which
was evident as early as 8 h following exposure of cells to A
,
which was correlated with increased apoptosis measured 48 h
following exposure to A
. In light of recent data showing that
activation of NF-
B in cells exposed to apoptotic stimuli suppresses
cell death (44-46, 48, 60), our findings suggest that the sustained
suppression of NF-
B activity plays a role in the proapoptotic action
of mutant PS-1. However, because the apoptotic process was enhanced in
cells expressing mutant PS-1, we cannot completely rule out the
possibility that the depressed NF-
B activity in cells expressing
mutant PS-1 was a consequence of early events in the apoptotic process.
Nevertheless, the ability of sAPP
to prevent both the delayed
suppression of NF-
B activity and apoptosis in cells expressing
mutant PS-1 and the ability of
B decoy DNA to block these effects of
sAPP
support a role for suppression of NF-
B activity in the
pathogenic mechanism of PS-1 mutations. The enhanced activation of
NF-
B during the first 4 h following exposure to A
in cells
expressing mutant PS-1 probably reflects the enhanced
[Ca2+]i and oxidative stress documented in our
calcium imaging and DCF fluorescence assays. Indeed, both calcium and
hydrogen peroxide are known to be potent activators of NF-
B in many
cell types including neurons (61-63). When taken together with the
data showing that
B decoy DNA enhanced A
-induced cell death, the enhanced short term activation of NF-
B in PC12 cells expressing mutant PS-1 is clearly not involved in the enhanced vulnerability of
these cells to apoptosis induced by A
and may represent a self-defense mechanism of the cells.
The gene targets of NF-
B that mediate cellular resistance to
apoptosis have not been firmly established. However, two candidates are
Mn-superoxide dismutase and the calcium-binding protein calbindin D28k.
Both Mn-superoxide dismutase and calbindin D28k are induced by TNF
and ceramide in cultured embryonic rat hippocampal neurons (48, 49),
and overexpression of calbindin D28k (64) and Mn-superoxide dismutase
(65) in cultured neurons protects them from being killed by oxidative
and metabolic apoptotic insults. Moreover, it was recently shown that
B decoy DNA blocks both induction of Mn-superoxide dismutase
expression and resistance to apoptosis in hippocampal neurons
treated with TNF
(48), strongly suggesting a role for this
antioxidant enzyme in the neuroprotective actions of NF-
B
activation. The evidence that A
toxicity in neurons involves both
oxidative stress and elevation of [Ca2+]i (31) is
consistent with a role for NF-
B-mediated regulation of expression of
genes involved in regulating calcium homeostasis and free radical
metabolism in the cytoprotective actions of sAPP
documented in the
present study.
The ability of dantrolene and nifedipine to protect PC12 cells against
the proapoptotic action of mutant PS-1 suggests pivotal roles of
calcium release from ER and influx through
voltage-dependent channels in the pathogenic mechanism of
PS-1 mutations. Consistent with this interpretation are recent data
showing that overexpression of the antiapoptotic gene product Bcl-2
stabilizes [Ca2+]i in PC12 cells expressing
mutant PS-1 following exposure to thapsigargin (28) or
A
.2 Bcl-2 also suppresses
oxidative stress and apoptosis induced by trophic factor withdrawal in
cells expressing mutant PS-1 (28). In nonneuronal cells, Bcl-2's
antiapoptotic actions have been linked to suppression of ER calcium
release (59). We found that the elevation of
[Ca2+]i and apoptosis induced by either A
or
thapsigargin was enhanced in cells expressing mutant PS-1. Our data
therefore suggest that aberrant ER calcium regulation plays a central
role in A
-induced apoptosis and that PS-1 mutations may promote
apoptosis by enhancing ER calcium release. sAPP
stabilized
[Ca2+]i and suppressed A
- and
thapsigargin-induced apoptosis in cells expressing mutant PS-1.
Stabilization of calcium homeostasis in cells treated with sAPP
was
correlated with maintenance of NF-
B activity and mitochondrial
function, consistent with the ability of well-known activators of
NF-
B such as TNF
to stabilize [Ca2+]i (46,
48).
Studies of brain tissue and cultured fibroblasts from AD patients have
provided evidence for perturbed calcium homeostasis in AD. Examples
include the following: 1) calcium-dependent protease activity is increased in degenerating neurons in AD brain tissue (66);
2) levels of acylphosphatase, an enzyme that modulates the activity of
the ER Ca2+-ATPase, are increased in fibroblasts from
patients bearing PS-1 mutations (67); 3) Ca2+ release from
ER in response to agonists linked to the inositol 1,4,5-trisphosphate
pathway is increased in fibroblasts from carriers of PS-1 mutations
(68); and 4) levels of inositol 1,4,5-trisphosphate binding to ER
membranes were reported to be decreased in cerebral cortical tissue
from sporadic AD patients (69). When taken together with data showing
altered levels of NF-
B immunoreactivity and activity in neurons in
vulnerable regions of AD patients (50, 51, 70), our findings suggest
mechanistic links between disruption of ER calcium homeostasis, NF-
B
activity, and neuronal apoptosis in AD. The present findings also
identify systems regulating ER calcium homeostasis (e.g.
inositol 1,4,5-trisphosphate receptors and Ca2+-ATPases),
NF-
B activity (e.g. upstream regulators, NF-
B
subunits, and NF-
B-responsive genes), and sAPP
production
(e.g. secretases) as potential targets for therapeutic
intervention in AD.
In addition to perturbed calcium homeostasis, accumulating data
strongly suggest major contributions of oxidative stress and mitochondrial dysfunction to the pathogenesis of neuronal degeneration in AD (for a review, see Refs. 71-73). Aberrant processing of APP may
promote neurodegeneration by increasing levels of neurotoxic forms of
A
, which induce oxidative stress and disrupt calcium homeostasis,
and/or by decreasing production of sAPP
, which exhibits neuroprotective activity (for a review, see Ref. 31). Our data suggest
that, by enhancing levels of oxidative stress and promoting mitochondrial dysfunction, PS-1 mutations may sensitize neurons to
A
-induced apoptosis. Levels of rhodamine 123 fluorescence and MTT
reduction (measures of mitochondrial transmembrane potential and energy
charge/redox state, respectively) were significantly decreased in PC12
cell lines expressing mutant PS-1 following exposure to A
compared
with vector-transfected lines and lines overexpressing wild-type PS-1.
In addition, levels of DCF fluorescence (a measure of cellular peroxide
levels) were significantly increased in cells exposed to A
, an
effect exacerbated by mutant PS-1. Activation of NF-
B appears to
play an important role in suppressing oxyradical production and
preserving mitochondrial function in cells exposed to A
, because
treatment of cells with
B decoy DNA enhanced the A
-induced
oxyradical accumulation and mitochondrial dysfunction and completely
blocked the protective effect of sAPP
. Oxyradical stress and
mitochondrial dysfunction in our paradigm may result from perturbed
cellular calcium homeostasis, because elevation of
[Ca2+]i induced by A
was correlated with
increased oxyradical levels and mitochondrial impairment and because
agents that block calcium release from ER (dantrolene) or influx
through voltage-dependent calcium channels (nifedipine)
suppressed oxyradical accumulation and mitochondrial dysfunction
(28).2 Moreover, sAPP
largely prevented A
-induced
elevations of [Ca2+]i and peroxide levels and
stabilized mitochondrial transmembrane potential in cells expressing
mutant PS-1; sAPP
also protected the cells against
thapsigargin-induced apoptosis. Collectively, the data suggest that
sAPP
prevents apoptosis by stabilizing calcium homeostasis,
suppressing oxyradical production, and preserving mitochondrial
function.
Additional mechanisms whereby presenilin mutations may promote
apoptosis are suggested by recent studies. For example, Kim and
co-workers (74) reported that PS-1 and PS-2 are cleaved by a caspase-3
family protease in a neuroglial cell line induced to undergo apoptosis.
Apoptosis induced by A
in neurons is blocked by inhibitors of
caspases, a family of proteins believed to play important roles in the
effector phase of apoptosis (64, 75). Caspase inhibitors also block
apoptosis in PC12 cells expressing mutant PS-1.2 However,
it is not known whether cleavage of PSs by caspases is mechanistically
involved in apoptosis or is a consequence of the apoptotic process.
Interactions of presenilins with APP have been reported (76, 77) and
might play indirect roles in promoting neuronal apoptosis.
Enzymatic processing of APP is altered in AD such that levels of A
production (particularly the A
-(1-42) form) are increased and
levels of sAPP
are decreased. Levels of A
-(1-42) production are
increased in cultured cells transfected with mutant presenilins, in
transgenic mice expressing mutant PS-1, and in cultured fibroblasts
from carriers of PS mutations (24, 78). It is unclear how presenilin
mutations alter APP processing, but one possibility is that the altered
processing is secondary to increased levels of cellular stress. In
support of this hypothesis, we have shown that PS-1 mutations
destabilize calcium homeostasis and enhance oxidative and metabolic
stress in PC12 cells (Refs. 27 and 28 and this study), and other laboratories have shown that sustained elevations of
[Ca2+]i (79) and metabolic/oxidative stress (80)
shift APP processing in favor of increased A
production. Prior
studies have shown that enzymatic processing of APP is altered in AD
such that there is reduced cleavage at the
-secretase site and
increased cleavage at the
- and
-secretase sites (for a review,
see Refs. 1 and 31). It is therefore likely that the increased A
production in cells expressing presenilin mutations is accompanied by
reduced levels of sAPP
, and, indeed, there is evidence that sAPP
levels are decreased in the cerebrospinal fluid of AD patients (81, 82), although data on sAPP
levels in carriers of presenilin mutations have not yet been reported. Because sAPP
is approximately 100-fold more potent than sAPP
in protecting cultured neurons against excitotoxic and oxidative insults (41), it is reasonable to
consider that decreased levels of sAPP
contribute to the
proapoptotic actions of mutant PS-1. Consistent with this possibility,
our data show that sAPP
is very effective in protecting PC12 cells expressing mutant PS-1 against apoptosis induced by A
.
We thank W. Fu, H. Luo, and R. Pelphrey for
technical assistance and K. Furukawa and I. Kruman for helpful
discussions.