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The Journal of Neuroscience, July 1, 1999, 19(13):5360-5369
Mutant Presenilin-1 Induces Apoptosis and Downregulates
Akt/PKB
Conrad C.
Weihl1,
Ghanashyam D.
Ghadge3,
Scott G.
Kennedy4,
Nissim
Hay4,
Richard J.
Miller1, 2, and
Raymond P.
Roos1, 3
1 Committee on Neurobiology and Departments of
2 Pharmacological and Physiological Sciences and
3 Neurology, University of Chicago, Chicago, Illinois
60637, and 4 Department of Molecular Genetics, The
University of Illinois at Chicago, Chicago, Illinois 60607
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ABSTRACT |
Most early onset cases of familial Alzheimer's disease (AD) are
caused by mutations in presenilin-1 (PS1) and presenilin-2 (PS2). These
mutations lead to increased  -amyloid formation and may induce
apoptosis in some model systems. Using primary cultured hippocampal
neurons (HNs) and rat pheochromocytoma (PC12) cells transiently
transfected with replication-defective recombinant adenoviral vectors
expressing wild-type or mutant PS1, we demonstrate that mutant PS1s
induce apoptosis, downregulate the survival factor Akt/PKB, and
affect several Akt/PKB downstream targets, including glycogen synthase
kinase-3 and -catenin. Expression of a constitutively active
Akt/PKB rescues HNs from mutant PS1-induced neuronal cell death,
suggesting a potential therapeutic target for AD. Downregulation of
Akt/PKB may be a mechanism by which mutant PS1 induces apoptosis and
may play a role in the pathogenesis of familial AD.
Key words:
presenilin; Alzheimer's disease; apoptosis; Akt/PKB; adenoviral vectors; hippocampal neurons
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INTRODUCTION |
Mutations in the presenilin genes
PS1 and PS2 cause most early onset familial Alzheimer's disease (AD)
(Price and Sisodia, 1998 ). These genes encode integral membrane
proteins of unknown function that are predicted to have six to eight
membrane-spanning domains (Price and Sisodia, 1998). The presenilins
facilitate -amyloid processing and are believed to play a role in
Notch signaling (Levitan et al., 1996; Scheuner et al., 1996). Several lines of evidence suggest that PS1 may also be involved in the Wnt
signaling pathway (Zhou et al., 1997; Murayama et al., 1998; Takashima
et al., 1998; Yu et al., 1998; Zhang et al., 1998; Nishimura et al.,
1999). Using yeast two-hybrid analysis, Zhou et al. (1997) identified
 -catenin, an armadillo repeat protein, as a putative partner
with PS1. Other studies have demonstrated that PS1 interacts in
vivo with -catenin and its regulatory kinase glycogen synthase kinase-3 (GSK-3) (Murayama et al., 1998; Takashima et al., 1998; Yu et al., 1998; Zhang et al., 1998; Nishimura et al., 1999). PS1 and
PS2 have also been reported to modulate apoptosis. Vito et al. (1996)
demonstrated that Alg-3, a cDNA with homology to PS2, rescues T
lymphocytes from apoptotic death. In addition, Wolozin et al. (1996)
showed that mutant PS2 transfection of rat pheochromocytoma (PC12)
cells enhanced basal levels of apoptosis over that seen after
PS2-wild-type (WT) transfection. Furthermore, PC12 cells that stably
express mutant PS1-L286V show an increased sensitivity to apoptosis
after trophic factor withdrawal and -amyloid administration (Guo et
al., 1996). However, other studies have failed to demonstrate that
mutant PS1 results in increased sensitivity to apoptotic stimuli
(Bursztajn et al., 1998).
Studies of mutant PS-induced apoptotic cell death have involved stably
transfected non-neuronal and neuroblastoma cell lines (Guo et al.,
1996; Wolozin et al., 1996). We investigated hippocampal neurons (HNs)
and PC12 cells transiently expressing PS1-WT or mutant PS1 using
adenoviral vectors (AdVs) to identify the effects of mutant PS1 on cell
viability and apoptosis. The advantages of this system are that the
cells studied, HNs, are prominently affected by AD, and that the use of
transient expression avoids selection of cell lines that may have
undergone mutation to avoid deleterious effects of mutant PS1
overexpression. Because PS1 may play a role in Wnt signal transduction,
and PS1 mutations may induce apoptosis, we questioned whether PS1 may
act at the point of convergence between these two signaling pathways.
We specifically investigated the effect of PS1 on Akt/PKB, a
serine/threonine kinase, and its downstream targets because Akt/PKB is
associated with antiapoptotic signaling (Dudek et al., 1997; Kennedy et
al., 1997) and is capable of inactivating GSK-3 (Cross et al.,
1995), a key component in Wnt signaling.
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MATERIALS AND METHODS |
Cell culture and reagents. PC12 cells were maintained
in DMEM supplemented with 10% bovine calf serum and 10 µg/ml
penicillin-streptomycin (Sigma, St. Louis, MO). HNs were prepared from
the hippocampi of fetal rats at 17 d of gestation, as previously
described (Ghadge et al., 1997). LY294002, a gift from Dr. Clive
Palfrey (University of Chicago), was diluted in DMSO and used at
10 µM.
Construction of AdVs and bicistronic expression plasmids.
AdPS1-WT, AdPS1-A246E, and AdPS1-C410Y were previously described (Weihl
et al., 1999). Bicistronic expression plasmids containing PS1-WT,
PS1-A246E, or PS1-C410Y cDNAs upstream of a LacZ reporter gene were constructed as follows: the encephalomyocarditis virus internal ribosomal entry site and -galactosidase cDNA
(LacZ) were digested from plasmid p1704LacZ and blunt
end-ligated into an EcoRV restriction site of pAdCMV
containing PS1-WT or mutant PS1.
Plasmid pAdKNmAkt/PKB contains the Akt/PKB cDNA fused to a
myristoylation sequence allowing for plasma membrane translocation. To construct AdmAkt/PKB, the plasmid pAdKNmAkt/PKB was linearized with
NheI and cotransfected, using the calcium phosphate
precipitation method, with XbaI- and
ClaI-digested adenovirus 5 (sub360) DNA into HEK293 cells, a
trans-complementing cell line for E1 function. The AdVs were
purified by CsCl isopycnic ultracentrifugation and then dialyzed
against HEPES-buffered saline to produce a high-titered virus stock.
AdV transgene expression was determined via Western analysis.
Recombinant virus infections and transfections. PC12 cells
were infected for 2 hr with an aliquot of purified virus to achieve a
multiplicity of infection (MOI) of 40 or 200 pfu/cell in a sufficient volume of postinfection media (culture media containing 2% serum) to
cover the cells, washed once, and then incubated with postinfection media. In the case of HNs, coverslips were removed from the glial feeder layer, and HNs were washed once in glial conditioned media and
then infected at an MOI of 1000 (except for AdmAkt when the MOI was
250) in conditioned media. Two hours later HNs were washed and replaced
inverted over the glial feeder layer.
DNA transfections were performed as previously described. In brief,
PC12 cells were plated in six-well culture plates, and 2 µg of the
respective expression plasmid was diluted in 100 µl of 0.15 M NaCl and 0.6 µl of 0.1 M polyethylenimine
(Aldrich, St. Louis, MO) and applied to wells containing 1.5 ml of
serum-free DMEM. The plates were spun in a clinical centrifuge for 10 min at 1000 rpm and then incubated for 2 hr, after which media were replaced with DMEM containing 5% serum. After 48 and 96 hr, the plates
were rinsed in PBS, fixed in 4% paraformaldehyde, and incubated for 4 hr at 37°C in
5-bromo-4-chloro-3-indolyl--D-galactopyranosidase (X-gal) staining solution (50 mM Tris-Cl, pH 7.5, 15 mM NaCl, 2 mM MgCl2, 0.5 mg/ml X-gal, and 2.5 mM ferriferrocyanide).
Immunohistochemistry and cell death assay. Coverslips were
rinsed in PBS, pH 7.4, fixed with ice-cold methanol for 10 min, and
blocked overnight in PBS containing 2% BSA and 0.1% Tween 20. Rat
monoclonal anti-PS1 antibody (Chemicon, Temecula, CA) diluted 1:200 in
blocking buffer was applied 1 hr at room temperature and rinsed
three times in blocking buffer. FITC-conjugated anti-rat IgG diluted
1:200 in blocking buffer was then applied 1 hr at room temperature and
rinsed three times in blocking buffer, and immunostaining was detected
via fluorescent microscopy. For nuclear staining, coverslips were
rinsed in PBS, pH 7.4, fixed 10 min in 4% paraformaldehyde, and then
stained with 50 µg/ml Hoescht 33342 (Molecular Probes, Eugene, OR).
The percentage of fragmented nuclei was determined using five
experiments and >10 fields per experiment.
MTT assay was performed as follows. One hundred microliters of 5 mg/ml
MTT (Sigma) in PBS were incubated per milliliter of culture media for 3 hr at 37°C. Precipitated MTT was resuspended in culture media with
the addition of 1 volume of acid isopropanol (0.04N HCl) and read on a
spectrophotometer at 570 nm.
Immunoprecipitation and Western blot. The monolayer was
rinsed in PBS, pH 7.4, scraped, and centrifuged. The cell pellet was resuspended in Laemmli buffer with 100 µg/ml phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml aprotinin, and 1 µg/ml leupeptin and sonicated on ice for 5 sec to shear chromosomal DNA. For the
preparation of cytosolic extracts, the cell pellet was resuspended in
lysis buffer (10 mM HEPES, pH 7.5, 10 mM KCl, 1 mM dithithreitol, and 1% NP-40) and centrifuged for 15 min
to remove cell debris. For immunoprecipitation, these lysates were
resuspended in PBS, precleared for 1 hr with protein A-Sepharose
(Pharmacia, Piscataway, NJ) and incubated overnight at 4.0°C with
primary antibody. The antigen-antibody complex was captured with
protein A-Sepharose and released by boiling in Laemmli buffer.
Sonicated or immunoprecipitated material was electrophoresed on a 10%
Tris-glycine-buffered SDS-polyacrylamide gel. The gel was
electroblotted to a 0.2 µm polyvinylidene difluoride membrane
(Schleicher & Schuell, Keene, NH) for 2 hr. The blot was blocked
overnight in PBS containing 0.1% Tween 20 and 5.0% milk and then
incubated at room temperature for 3 hr with primary antibody, rinsed
three times for 15 min, and incubated for 1 hr in peroxidase-conjugated
secondary antibody diluted in PBS containing 0.05% Tween 20. Detection
was performed using ECL Plus and Hyperfilm ECL (Amersham, Arlington
Heights, IL).
The following antibodies were used: anti-PS1loop (Thinakaran et al.,
1996), anti-GSK-3 (QCB, Hopkinton, MA), anti-GSK-3(Ser9) (QCB),
anti-GSK-(Tyr216) (Upstate Biotechnology, Lake Placid, NY), anti-p85
(Upstate), anti-phosphotyrosine (Upstate), and anti--catenin (Transduction Laboratories, Lexington, KY). Phospho-Akt(Ser473), phospho-stress-activated protein kinase (SAPK)/c-Jun N-terminal protein
kinase (JNK)(Thr183/Tyr185), phospho-p38 MAP kinase(Thr180/Tyr182), phospho-p44/42 extracellular signal-regulated kinase (ERK1/2) kinase(Thr202/Tyr204), and phospho-p70S6 kinase(Ser411) antibody kits
are from New England Biolabs (Beverly, MA). All dilutions were
according to manufacturers' instructions.
Akt/PKB kinase assay. Cells from transfected 100 mm plates
were lysed in buffer A (50 mM Tris-Cl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM
Na3VO4, 0.1% -mercaptoethanol, 1%
Triton X-100, 50 mM NaF, 5 mM sodium
pyrophospate, 10 mM sodium -glycerophosphate, 0.1 mM PMSF, and 1 µg/ml aprotinin and leupeptin) and
immunoprecipitated with 4 µg of anti-Akt/PKB PH domain antibody
(Upstate) prebound to protein A/G-agarose (Santa Cruz Biotechnology,
Santa Cruz, CA) for 90 min at 4.0°C. Immunoprecipitated material was
washed three times in buffer A containing 0.5 M NaCl and
two times in buffer B (100 mM 4-morpholinepropanesulfonic
acid, pH 7.2, 125 mM -glycerophosphate, 25 mM EGTA, 5 mM
Na3VO4, and 5 mM DTT). The
immune complex was incubated in 50 µl of buffer B, with 10 µM inhibitor peptide (Upstate), 100 µM
Akt/PKB-specific substrate (Upstate), and 10 µCi of
[ -33P]ATP at room temperature for 10 min. The reaction
was stopped with addition of 40% TCA for 5 min, and 40 µl of the
reaction mix was then transfered to P81 phosphocellulose paper and
washed three times with 50 ml of 0.75% phosphoric acid. Samples were transfered to scintillation tubes with 5 ml of scintillation mixture and counted in a scintillation counter.
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RESULTS |
Mutant PS1 expression induces apoptosis in HNs and PC12 cells
HNs were transduced with AdVs that expressed PS1-WT or one of two
mutants associated with familial AD, PS1-A246E and PS1-C410Y. Using a
sensitive monoclonal antibody directed against the N terminus of human
PS1, we were able to specifically immunostain HNs that expressed human
PS1, because the antibody does not cross-react with rodent PS1.
Immunofluorescent studies showed that ~50% of HNs carried the human
PS1 transgene 24 hr after transduction. Human PS1 localized in a
perinuclear distribution and in both axonal and dendritic processes
(Fig. 1a), indicating that
overexpression of PS1 results in a localization similar to that
described for endogenous PS1 (Cook et al., 1996; Lah et al., 1997).
Confocal microscopy showed that PS1-WT and mutant PS1 had a similar
localization (data not shown). The intensity of the immunofluorescent
staining as well as Western analysis of lysates from adenovirally
transduced cells 72 hr after transduction demonstrated that PS1 protein
expression levels of PS1-WT- and mutant PS1-expressing cells were
similar (Fig. 1b). Of note is a 14 kDa PS1-immunoreactive
fragment present in lysates from PS1-A246E- and PS1-C410Y-expressing
cells (Fig. 1b). This fragment coincides with a previously
reported putative PS1 caspase-cleaved fragment (Loetscher et al., 1997;
Weihl et al., 1999).
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Figure 1.
AdV epression of PS1 in neurons.
a, Immunohistochemistry of HNs with a rat monoclonal
antibody specific for human PS1 24 hr after transduction with PS1-WT or
the indicated PS1-mutant. b, Western analysis using
anti-PS1loop antibody on lysates of PC12 cells 36 hr after
transduction. The AdVs used in the transduction of cells processed for
this and subsequent Western blots are noted above the lanes. The
full-length holoprotein (HP), processed C-terminal
fragment (CTF) and caspase-cleaved CTF
(CTFcasp) are noted.
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We used Hoescht 33342 fluorescent staining to determine whether mutant
PS1 induced apoptosis of HNs. The staining demonstrated that the number
of fragmented apoptotic nuclei was greatly increased in HNs expressing
mutant PS1s when compared with those expressing PS1-WT or a control
gene (LacZ) or when compared with mock-transduced neurons
(Fig. 2a). The number of
fragmented nuclei peaked at 5 d after transduction (Fig.
2b). Additional staining for human PS1 expression showed
that it was the mutant PS1-expressing HNs that exhibited fragmented
nuclei, whereas HNs expressing PS1-WT had intact nuclei (Fig.
2c). Similar results were obtained after PC12 cell
transduction with mutant PS1s using Hoescht 33342 nuclear staining
(data not shown). To quantify this data by an alternative method, we
used a colormetric assay based on the tetrazolium salt MTT, which
measures only living cells (Fig. 2d).
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Figure 2.
Mutant PS1 expression induces apoptosis in HNs and
PC12 cells. a, Hoescht 33342 nuclear staining of HNs
3 d (top panel) or 5 d (bottom
panel) after transduction with AdLacZ, AdPS1-WT, or the
indicated AdPS1-mutant. Arrowheads show fragmented
nuclei. b, Percentage of fragmented nuclei from HNs 3 and 5 d after transduction with mock, LacZ, PS1-WT, or mutant PS1.
**p < 0.005 for PS1-mutant when compared with
control transduced HNs from five separate experiments.
c, Double fluorescent staining of HNs 4 d after
transduction with PS1-WT or the indicated PS1-mutant using a rat
monoclonal antibody for human PS1 and Hoescht 33342 for nuclear
morphology. d, Ratio of MTT production from PC12 cells 4 or 6 d after transduction with AdPS1-WT or the indicated
AdPS1-mutant compared with mock controls at each time point, which was
arbitrarily set to 1. **p < 0.005 for PS1-mutant
when compared with control transduced PC12 cells from three separate
experiments. e, Ratio of the number of LacZ-positive
cells 48 or 96 hr after transient transfection of a bicistronic
expression vector containing LacZ in the second cistron preceded by
PS1-WT or PS1-mutant cDNAs in the first cistron compared with the
number LacZ-positive cells 24 hr after transfection of a monocistronic
LacZ construct. *p < 0.05; **p < 0.005 for PS1 mutant when compared with monocistronic
LacZ-transfected PC12 cells from four separate experiments.
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To confirm that mutant PS1-associated cell death was unrelated to the
use of adenoviral vectors, we transiently transfected PC12 cells with
bicistronic expression vectors expressing LacZ downstream of PS1-WT,
PS1-A246E, or PS1-C410Y cDNAs. Forty-eight and 96 hr after transfection
the number of LacZ-positive cells was counted. PS1-mutant-transfected
cells demonstrated significantly fewer LacZ-positive cells at both time
points when compared with PS1-WT or a LacZ monocistronic plasmid
control, suggesting that transient transfection of mutant PS1 decreases
cell survival (Fig. 2e). These studies confirm that mutant
PS1 expression induced the nuclear condensation and neuronal apoptosis.
Downregulation of Akt/PKB activity is an early step in
PS1-mutant-associated apoptosis
Lysates of HNs and naive (undifferentiated) PC12 cells 36 hr after
PS1-WT or mutant PS1 transduction were Western blotted using an
antibody specific for the phosphorylated, active form of Akt/PKB. No
morphological signs of apoptosis or cell death were apparent with
mutant PS1-transduced HNs and PC12 cells at this time point, suggesting
that any change in Akt/PKB activity would be an early step in mutant
PS1-induced neuronal death. HNs and naive PC12 cells transduced with
mutant PS1s demonstrated a 25-50% decrease in basal levels of Akt/PKB
phosphorylation (Fig. 3a-c)
when compared with lysates from HNs or PC12 cells expressing PS1-WT or
a control [wild-type superoxide dismutase (SOD); (Ghadge et al.,
1997)] or with lysates from mock-transduced cells. A similar decrease
was seen in naive PC12 cells that were serum starved for 1 hr and
stimulated with nerve growth factor (NGF) for 10 min (Fig.
3a,c). In this experiment and all subsequent experiments, NGF stimulation refers to a brief 10 min application of NGF to serum-starved naive PC12 cells before cell lysis. The increase in
Akt/PKB phosphorylation after SOD expression is a result of AdV
infection, because transduction with an AdV expressing LacZ demonstrated a similar increase (data not shown).
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Figure 3.
Mutant PS1 expression downregulates
Akt/PKB. a, Western analysis using anti-Akt/PKB
(top panel) or anti-phospho-Akt/PKB (Ser473)
antibody (AKTpS) (bottom panel) on lysates of
PC12 cells 36 hr after transduction with (right
panel) and without (left panel) 10 min NGF stimulation. b, Western analysis using
anti-Akt/PKB (top panel) or
anti-phospho-Akt/PKB(Ser473) antibody (bottom
panel) on lysates of HNs 36 hr after transduction.
c, Densitometric analysis of the ratio of
phospho-Akt/ PKB(Ser473) to total Akt/PKB from four separate
experiments. **p < 0.005 for PS1 mutant when
compared with controls. In calculating this ratio and ratios in
subsequent figures, the mock was arbitrarily set to 1 for each
condition. d, In vitro kinase assay of
PC12 cell lysates 36 hr after transduction with and without 10 min NGF
stimulation. *p < 0.05; **p < 0.005 for PS1-mutants when compared with PS1-WT are from more than four
separate experiments.
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Although the phosphorylation state of Akt/PKB is a good indicator of
its activity, we chose to further confirm downregulation of Akt/PKB
after mutant PS1 expression using an in vitro kinase assay.
Immunoprecipitated Akt/PKB from PC12 cells expressing PS1-WT, PS1-A246E, or PS1-C410Y was assayed for the transfer of radiolabeled ATP to an Akt/PKB-specific substrate peptide. Figure 3d
shows that Akt/PKB activity was reduced by 40% in PS1-A246E-expressing cell lysates and by 65% in PS1-C410Y-expressing cell lysates when compared with PS1-WT-expressing cell lysates. NGF stimulation for 10 min modestly increased Akt/PKB activity in both PS1-WT- and
PS1-mutant-expressing cells.
Mutant PS1-associated downregulation of Akt/PKB is not associated
with a perturbation of known upstream kinase pathways
Akt/PKB is phosphorylated through a cascade of kinases that are
stimulated by NGF. More specifically, NGF stimulation leads to TrkA
receptor autophosphorylation, which in turn recruits the p85 regulatory
subunit of phosphoinositide-3 kinase (PI3K) to the plasma membrane. At
the plasma membrane, p85 is phosphorylated by both TrkA and p110, the
catalytic subunit of PI3K, which in turn phosphorylates lipid
messengers that signal for Akt/PKB activation (Coffer et al., 1998). To
determine whether NGF and TrkA signaling was perturbed in mutant
PS1-expressing neurons and contributed to the observed decrease in
basal and NGF-stimulated Akt/PKB activity, we examined other kinase
pathways associated with this trophic factor signaling. Western blots
of PC12 cells 36 hr after transduction with mutant PS1s showed no
difference, when compared with control lysates, in basal or
NGF-stimulated phosphorylation of TrkA or in global tyrosine
phosphorylation (Fig. 4a).
There blots and subsequent blots are representative of at least three
experiments; hence minor inconsistencies present in the representative
blots were not seen with all experiments. When possible, we have
densitometrically quantified the results of separate experiments and
the data are shown when relevant. We assessed the activation of PI3K by
immunoprecipitating similar NGF-stimulated PC12 lysates with an
antibody specific for phosphorylated tyrosines and subsequently
immunoblotting with an antibody directed to the p85 subunit of PI3K.
The phosphorylation of p85 was unchanged after mutant PS1 expression
compared with lysates from cells expressing PS1-WT or controls,
suggesting that TrkA activation of PI3K is unaffected (Fig.
4b). As a negative control we immunoprecipitated
unstimulated PC12 cell lysates [Fig. 4b,
mock( NGF)]. In addition, TrkA activation of
MAP kinases ERK1 and ERK2 was unaffected after mutant PS1 expression
(Fig. 4c). These results indicate that the trophic factor
signaling necessary for Akt/PKB phosphorylation and concomitant
activation is intact and unchanged in mutant PS1-expressing
neurons.
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Figure 4.
Mutant PS1 expression does not affect other kinase
pathways. All blots are representative of at least three independent
experiments. a, Western analysis using
anti-phosphotyrosine antibody on lysates of PC12 cells 36 hr after
transduction with (right panel) and without
(left panel) 10 min NGF stimulation.
b, Densitometric analysis of the amount of
tyrosine-phosphorylated p85 from three western blots of transduced PC12
cell lysates 36 hr after transduction following a 10 min NGF
stimulation (which were immunoprecipitated with anti-phosphotyrosine
antibody before electrophoresis) immunostained with anti-p85 antibody.
Mock(NGF), Negative control for NGF
stimulation. c, Western analysis using anti-p44/42
ERK1/2 kinase antibody (top panel) or
anti-phospho-p44/42 ERK1/2 kinase(Thr202/Tyr204) antibody
(bottom panel) on lysates of PC12 cells 36 hr
after transduction with (right panel) and without
(left panel) 10 min NGF stimulation. The
Mock lane is overexposed to allow a comparison between
the PS1-WT and mutant PS1 lanes.
d, Western analysis using anti-SAPK/JNK antibody
(top panel) or
anti-phospho-SAPK/JNK(Thr183/Tyr185) antibody (bottom
panel) on lysates of PC12 cells 36 hr after transduction
with (right panel) and without (left
panel) 10 min NGF stimulation. e, Western
analysis using anti-p38 MAP kinase antibody (top
panel) or anti-phospho-p38 MAP kinase(Thr180/Tyr182)
antibody (bottom panel) on lysates of PC12 cells
36 hr after transduction with (right panel) and
without (left panel) 10 min NGF
stimulation.
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Mutant PS1-induced apoptosis is not related to stress-activated
kinase pathways
We wondered whether other kinase pathways unassociated with the
direct regulation of Akt/PKB activity mediated changes in neurons
induced by mutant PS1 expression. The SAPKs, for example, have been
shown to be activated by a variety of cellular stresses and apoptotic
stimuli and may indirectly regulate Akt/PKB activity (Berra et al.,
1998; Zundel and Giaccia, 1998). We found no differences in the
phosphorylation states of SAPK/JNK or p38/high-osmolarity glycerol
response (HOG) stress-related kinases in Western blots of
lysates of PC12 cells after transduction with mutant PS1s compared with
PS1-WT (Fig. 4d,e). The increase in phospho-SAPK/JNK seen in
all groups after AdV transduction is consistent with results from
previous reports using AdV infection (See and Shi, 1998). These results
indicate that the observed differences in apoptosis or Akt/PKB activity
after mutant PS1 expression are not a result of cell signaling through
stress-activated kinase pathways.
Mutant PS1-associated apoptosis affects the ribosomal
S6 kinase
Akt/PKB affects several aspects of cellular metabolism, in
particular, protein synthesis, glycogen metabolism, cell cycle regulation, cell differentiation, and cell survival (Coffer et al.,
1998). We examined several of these putative downstream targets in PC12
cells expressing PS1-WT or mutant PS1. The ribosomal S6 kinase
(p70S6k), which changes the pattern of protein synthesis after
mitogenic stimulation of cells, lies downstream of Akt/PKB and is
phosphorylated by a number of kinases (Coffer et al., 1998). Western
analysis of lysates from PC12 cells transduced with mutant PS1s
demonstrated a decrease in active p70S6k when compared with lysates
from PC12 cells expressing PS1-WT or SOD or with lysates from
mock-transduced PC12 cells, consistent with its activation through an
Akt/PKB signaling pathway (Fig.
5a,b). NGF stimulation of
mutant PS1-transduced cells boosted phosphorylated p70S6k to mock
levels (Fig. 5a,b), suggesting that basal levels of p70S6k activity may be mediated through Akt/PKB and that the other signaling pathways necessary to activate p70S6k via NGF are intact. It is interesting to note that NGF-stimulated activation of p70S6k occurs through a wortmannin-sensitive PI3K pathway and that p70S6k is phosphorylated by phosphoinositide-dependent protein kinase 1 (PDK1),
the same kinase responsible for Akt/PKB activation (Alessi et al.,
1998; Pullen et al., 1998). Because NGF-stimulated p70S6k phosphorylation is normal in mutant PS1-expressing cells, and because
p70S6k uses a pathway identical to that involved in NGF-stimulated Akt/PKB activity, we suggest that mutant PS1-associated basal and
NGF-stimulated downregulation of Akt/PKB in HNs is a specific defect
and not secondary to a more general abnormality in this pathway.
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Figure 5.
Mutant PS1-induced apoptosis is associated with
pathways downstream of Akt/PKB. a, Western analysis
using anti-p70S6k (top panel) or
anti-phospho-p70S6k(Ser411) antibody (bottom
panel) on lysates of PC12 cells 36 hr after transduction
with (right panel) and without (left
panel) 10 min NGF stimulation. b,
Densitometric analysis of the ratio of phospho-p70S6k(Ser411) to total
p70S6k from four separate experiments. **p < 0.005 for PS1 mutant when compared with controls.
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Mutant PS1-associated apoptosis affects GSK-3
and -catenin
Considerable attention has recently been focused on the role of
PS1 in the Wnt signaling pathway. It has been shown that PS1 associates
with -catenin and its regulatory kinase, GSK-3 (Zhou et al.,
1997; Murayama et al., 1998; Takashima et al., 1998; Yu et al., 1998;
Zhang et al., 1998). Wnt signaling occurs through activation of the Frz
receptor, which signals the inactivation of GSK-3, resulting in
increased -catenin stability (Cadigan and Nusse, 1997). This allows
for -catenin to enter the nucleus and activate the T-cell
factor/lymphoid-enhancing factor-1 (Tcf/LEF-1) family of transcription
factors (Cadigan and Nusse, 1997). A complementary pathway involves
trophic factor stimulation of Akt/PKB, resulting in phosphorylation of
GSK-3 on serine residue 9, leading to its inactivation (Cross et
al., 1995). We surmised that a decrease in Akt/PKB activity associated
with mutant PS1 expression in neurons would lead to decreased
inactivation (and enhanced activity) of GSK-3. Immunoprecipitation
of GSK-3 and subsequent immunostaining with an antibody specific for
phophoserine-9 of GSK-3 demonstrated that mutant PS1-expressing PC12
cells failed to phosphorylate GSK-3 as efficiently as cells
expressing PS1-WT or SOD or mock-transduced cells (Fig.
6a). In agreement with these
data, immunoblots of the same lysates with an antibody against
phosphotyrosine-216 of (activated) GSK-3 demonstrated that mutant
PS1-expressing neurons had an increase in phosphotyrosine-216 GSK-3
immunoreactivity when compared with PS1-WT-, SOD-, or mock-transduced
control cells (Fig. 6b). NGF stimulation for 10 min did not
significantly alter the phosphorylation of GSK-3 in PS1-mutant
expressing cells (data not shown). These data confirm that the mutant
PS1-associated decrease in Akt/PKB activity results in decreased
inactivation of GSK-3 with a subsequent enhancement in activity.
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Figure 6.
Mutant PS1-associated apoptosis
modulates components of Wnt signaling. a,
Immunoprecipitation with anti-GSK-3 followed by Western analysis
with anti-GSK-3 (top panel) or
anti-phospho-GSK-3(Ser9) antibody (bottom
panel) on lysates of PC12 cells 36 hr after
transduction. b, Western analysis using anti-GSK-3
(top panel) or anti-phospho-GSK-3(Tyr216)
antibody (bottom panel) on lysates of PC12 cells
36 hr after transduction. c, Western analysis from two
separate experiments using anti--catenin antibody on cytosolic
lysates of PC12 cells 36 hr after transduction. Loading controls show
immunoblotted anti-Akt/PKB antibody from the same lysates.
d, Densitometry of -catenin normalized to total
protein concentration from three separate experiments.
**p < 0.005 for PS1 mutant when compared with
controls.
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GSK-3 has many targets, including glycogen synthase, tau, and
-catenin (Plyte et al., 1992). To examine the physiological consequence of increased GSK-3 activity, we assessed the levels of
soluble -catenin, because phosphorylation of -catenin targets it
for proteosomal degradation (Plyte et al., 1992). Immunoblots of PC12
cytosolic lysates with an antibody for -catenin demonstrated a
decrease in the amount of -catenin in cells expressing PS1-WT and
mutant PS1s when compared with SOD- or mock-transduced cells (Fig.
6c,d). The decrease in mutant PS1-transduced cells was
greater than that seen in PS1-WT-transduced cells, suggesting that
mutant PS1-associated upregulation of GSK-3 results in a decrease in -catenin levels.
Mutant PS1-induced apoptosis is sensitive to PI3K inhibition and is
rescued by a constitutively active Akt/PKB
If mutant PS1-induced apoptosis is mediated through Akt/PKB
signaling, we reasoned that inhibiting Akt/PKB phosphorylation should
induce apoptosis in HNs and increase the apoptosis associated with
mutant PS1 expression. To test this, we used LY294002, a potent PI3K
inhibitor that has been shown to decrease Akt/PKB phosphorylation and
activity in vivo (Coffer et al., 1998). After treatment with
LY294002, the levels of apoptosis were elevated in PS1-WT- and
SOD-expressing HNs and highest after mutant PS1 expression (Fig.
7a). Similarly, we tested
whether expression of a constitutively active Akt/PKB could rescue HNs
from mutant PS1-induced apoptosis. To express a constitutively active
Akt/PKB, we prepared an AdV that expresses a myristoylated form of
Akt/PKB (AdmAkt/PKB), because Akt/PKB must move to the plasma membrane before it is phosphorylated and becomes active (Alessi and Cohen, 1998). Figure 7b demonstrates the increase in total as well
as phosphorylated Akt/PKB after transduction of PC12 cells with
AdmAkt/PKB compared with mock and AdSODWT controls. Co-transduction of
HNs with both mAkt/PKB and mutant PS1s resulted in complete rescue from
the PS1-mutant-induced HN apoptosis at 3 d after transduction and
partial rescue at 5 d after transduction (Fig. 7c).
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[in a new window]
|
Figure 7.
Mutant PS1-associated apoptosis is
increased by inhibition of Akt/PKB and rescued by constitutively active
Akt/PKB. a, c, Percentage of fragmented nuclei in HNs
3 d after transduction with mock, SOD, PS1-WT, or mutant PS1 along
with co-treatment with LY294002 (a) or
co-transduction with AdmAkt/PKB (c), and 5 d
after co-transduction with mutant PS1 along with AdmAkt/PKB
(c). b, Western analysis using
anti-Akt/PKB or anti-phospho-Akt/PKB(Ser473) antibody on lysates of
PC12 cells 36 hr after transduction.
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| |
DISCUSSION |
In summary, we have demonstrated that expression of familial
AD-associated mutant PS1s in HNs and PC12 cells results in apoptotic cell death. Our results are the first to show a direct proapoptotic effect of mutant PS1 in HNs, the target cell of AD pathogenesis. This
death is accompanied by an early decrease in survival signaling through
the Akt/PKB pathway. The decrease in Akt/PKB activity affects several
proteins associated with cell metabolism and cell survival: the basal
activity of the translational regulator p70S6k is reduced;
Akt/PKB-mediated inactivation of GSK-3 is reduced, resulting in an
increase in the activity of GSK-3; and the levels of soluble
-catenin are decreased. Mutant PS1-expressing HNs have increased
cell death after Akt/PKB inhibition, whereas expression of a
constitutively active form of Akt/PKB rescues HNs from mutant PS1-induced cell death. The latter observations may have therapeutic implications in AD.
We considered whether downregulation of Akt/PKB after expression of
mutant PS1 is a direct effect or merely a consequence of the
accompanying apoptosis. Recent evidence suggests that downregulation of
Akt/PKB activity can occur through mechanisms associated with some
forms of apoptosis. For example, ceramide-induced apoptosis in some
cells has been shown to be associated with a decrease in Akt/PKB
activity (Zhou et al., 1998; Zundel and Giaccia, 1998). In addition, an
increase in phosphatase activity present during apoptosis can result in
an increase in Akt/PKB inactivation (Meier et al., 1998).
Despite these reports, we doubt that the decrease in Akt/PKB activity,
as well as the increase in GSK-3 activity and the decrease in
-catenin levels, are merely a result of apoptosis. Of importance is
the fact that these biochemical changes occur 36 hr after transduction,
well before any morphological signs of apoptosis, which peak at 5 d after transduction. In addition, we found that PS1-mutant-expressing
stably transfected neuroblastoma cells, which do not show an enhanced
level of basal apoptosis, show a decrease in Akt/PKB activity (data not
shown), demonstrating that the decline in Akt/PKB is not a result of
apoptosis. Our data are supported by several reports demonstrating an
increase in apoptosis after PS1-mutant expression (Guo et al., 1996;
Wolozin et al., 1998). In addition, other groups have found that mutant PS1-expressing cells have a decrease in the stability or trafficking of
-catenin and an increase in GSK-3 activity in cells that were not
undergoing apoptosis (Murayama et al., 1998; Takashima et al., 1998;
Zhang et al., 1998; Nishimura et al., 1999), suggesting that our
findings are not an artifact of apoptosis.
The question remains how PS1, an integral membrane and endoplasmic
reticulum- and Golgi-associated protein, mediates the activity of
Akt/PKB. Recent evidence suggests that PS1 associates with GSK-3
(Takashima et al., 1998) and -catenin (Zhou et al., 1997; Murayama
et al., 1998; Yu et al., 1998; Zhang et al., 1998; Nishimura et al.,
1999). Because GSK-3 is known to associate with Akt/PKB (van Weeren
et al., 1998), it may be that PS1 associates with GSK-3,
-catenin, and Akt/PKB in a large complex. It is intriguing to note
that Akt/PKB must move from an intracellular location to the plasma
membrane before becoming activated by PDK1 (Alessi and Cohen, 1998) and
that constitutively active mutants of Akt/PKB target the membrane
facilitating this transit (Alessi and Cohen, 1998). Perhaps PS1 is
responsible for this trafficking step, whereas mutant PS1 disrupts the
trafficking, resulting in a decreased activation of Akt/PKB.
Mutant PS1 could result in an increased sensitivity to apoptotic
stimuli by several mechanisms, including a decrease in Akt/PKB survival
signaling, upregulation of GSK-3, or a decrease in
-catenin-associated transcriptional activation. Akt/PKB has a clear
role in some cell survival paradigms, because expression of
dominant-negative constructs leads to an increase in apoptotic cell
death, whereas overexpression of constitutively active Akt/PKB rescues
some cells from a variety of apoptotic stimuli (Kennedy et al., 1997).
Upregulation of GSK-3 may also mediate apoptosis. Its constitutive
expression results in increased basal levels of apoptosis in PC12 cells
(Pap and Cooper, 1998). Finally, a recent report demonstrates that
expression of a dominant negative -catenin mediates apoptosis in
cortical neurons (Zhang et al., 1998).
Although compelling evidence links PS mutations to abnormal amyloid
precursor processing and -amyloid deposition (Scheuner et al.,
1996), little is known about how PS mutations result in the
hyperphosphorylation of microtuble-associated protein tau, a
pathological hallmark of AD. Interestingly, GSK-3 phosphorylates tau
as well as -catenin (Lovestone et al., 1994). Therefore, the
increased activity of GSK-3 that we found after mutant PS1 expression may lead to tau hyperphosphorylation. Our observations are
consistent with reports that AD brain tissues have increased levels of
GSK-3 and that inhibition of GSK-3 reduces tau phosphorylation in
neurons (Hong et al., 1997; Pei et al., 1997). Therefore, the decrease
in Akt/PKB-mediated inactivation of GSK-3 associated with mutant PS1
expression may play a central and critical role in AD pathogenesis by
increasing the hyperphosphorylation of tau, increasing neuronal
apoptosis, and altering amyloid precursor protein processing.
| |
FOOTNOTES |
Received Feb. 23, 1999; revised April 5, 1999; accepted April 14, 1999.
C.C.W. was supported by US Public Health Service Grant F30MH11697. We
thank Drs. S. Sisodia and G. Thinakaran for reagents and advice in the
preparation of this manuscript, Dr. J. Schaak for AdLacZ, and C. P. Mauer for culturing hippocampal neurons. Special thanks to Dr. V. P. Bindokas for help with the cover illustration.
Correspondence should be addressed to Dr. Raymond P. Roos, University
of Chicago Medical Center, Department of Neurology MC2030, 5841 S. Ellis Avenue, Chicago, Illinois 60637.
| |
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TOP
ABSTRACT
INTRODUCTION
