From the
Department of Pharmacology, Biocenter,
University of Frankfurt, 60439 Frankfurt, Germany and the
Department of Biochemistry, Adolf Butenandt
Institute, 80336 Munich, Germany
Received for publication, December 3, 2002 , and in revised form, April 23, 2003.
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ABSTRACT |
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INTRODUCTION |
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Several mutations in the APP and presenilin genes cause some forms of
familial Alzheimer's disease (FAD)
(79).
These mutations alter APP processing with respect to an enhanced A
production (10,
11) and have been associated
with an increased vulnerability to cell death
(1215).
-Secretase cleavage of the Swedish double mutation form of APP (APPsw;
K670M/N671L) occurs in Golgi-derived secretory vesicles, whereas wild-type APP
(APPwt) must be reinternalized before
-secretase cleavage
(16). This altered APP
production leads to a 36-fold increased A
production of both
A
-(140) and A
-(142)
(17,
18), which probably possess
the same structural peptide architecture
(19,
20). Recent reports have
demonstrated an intraneuronal A
accumulation in transgenic mice
expressing FAD proteins (21).
Alzheimer mice transgenic for APPsw mutation, but not wild-type mice,
exhibited an age-dependent increase in soluble A
-(140) and
A
-(142) levels and progressive amyloid deposition in brain
(22,
23).
Despite all of these findings, the underlying mechanisms responsible for the massive neurodegeneration in early age of FAD patients are still not completely understood. Persuasive evidence indicates that oxidative stress plays an important role in the neuropathological process in AD (2427). AD-related mutations probably enhance oxidative stress. Increased oxidative stress levels have been found in the temporal inferior cortex from Swedish FAD patients (28), and brains of mice transgenic for human presenilin 1 show reduced antioxidative enzyme activity (29).
Moreover, convincing evidence indicates that A is neurotoxic,
probably via an apoptotic pathway
(12,
3034).
A
is believed to play a major role in promoting neuronal degeneration
and death by rendering neurons vulnerable to age-related increases in levels
of oxidative stress and impairments in cellular energy metabolism
(32,
35). New evidence indicates
that reactive oxygen species-induced cellular events implicate the activation
of mitogen-activated protein kinases
(36,
37). Oxidative stress may
cause the activation of c-Jun N-terminal kinase (JNK; also known as
stress-activated protein kinase) in degenerating neurons in AD
(38). An involvement of JNK
pathway by the induction of Fas ligand in A
-induced neuronal apoptosis
was described by Morishima et al.
(39). Activation of JNK and
p38 associated with amyloid deposition was observed in mice transgenic for the
human APPsw mutation (40). The
inhibition of the JNK pathway was proposed as a potential therapeutic target
in AD (41).
Caspases can be divided into initiator caspases and effector caspases based on the presence of a large prodomain at their amino-terminal region. Initiator caspases containing a long prodomain, like caspase 2, caspase 8, caspase 9, and caspase 10 generally act in early stages of a proteolytic cascade, whereas effector caspases, like caspase 3, caspase 6, and caspase 7 act downstream and are involved in the cleavage of specific cellular proteins (for a review, see Refs. 42 and 43). Among the identified caspases, caspase 3 is of particular interest, since it appears to be very important in the progression of AD (12, 44, 45). In addition to caspase 3, other caspases have been associated with AD (4649).
Here we report on the effects of FAD-related APP mutation, using stably
transfected PC12 (rat pheochromocytoma) cells expressing the Swedish mutant
APP, wild-type APP, or empty vector. We examined the effect of hydrogen
peroxide on the activation of different caspases and the JNK pathway to
elucidate the relationship between mutant APP expression, increased A
production, and neuronal cell death. Furthermore, we investigated the
protective effects of caspase inhibitors as well as JNK inhibitor in
preventing oxidative stress-mediated cell death.
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EXPERIMENTAL PROCEDURES |
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Rat-specific caspase 3 and caspase 9 antibodies were also purchased from Cell Signaling Technology. Caspase 2 antibody was purchased from Alexis. Caspase 8 antibody was from Biocat. The caspase substrates were purchased from Calbiochem (Ac-DEVD-para-nitroaniline (pNA), Ac-VEID-pNA, Ac-VDVAD-pNA, and Ac-IETD-pNA for caspase 3, caspase 6, caspase 2, and caspase 8, respectively). JNK inhibitor II (SP600125; anthrax(1,9-cd)pyrazol-6(2H)-one) was from Calbiochem.
Cell Culture and TransfectionThe generation and
characterization of PC12 cell lines expressing human APPwt-, mutant APP
(APPsw)-, and vector-transfected control clones have been described previously
(12). In brief, PC12 cells
were transfected with DNA constructs harboring human mutant (APPsw;
K670M/N671L) or wild-type APP (APPwt) gene, inserted downstream of a
cytomegalovirus promotor, using the FUGENE technique (Roche Applied Science).
The transfected cell lines APPwt PC12, APPsw PC12, and vector-transfected PC12
were cultured in Dulbecco's modified Eagle's medium supplemented with 10%
heat-inactivated fetal calf serum, 5% heat-inactivated horse serum, 50
units/ml penicillin, 50 µg/ml streptomycin, and 400 µg/ml G418 at 37
°C in a humidified incubator containing 5% CO2. The stably
transfected clones (APPwt M5, N10, and U7 and APPsw Q8 and Q9) were selected
for the present study based on their similar expression of APP695 and the
5-fold increased secretion of A-(140) in the APPsw clones
(Fig. 1). To rule out potential
influences on the APP expression levels, APPsw and APPwt clones were routinely
screened in parallel to the experiments presented in this work. No alterations
in the expression of APP were found.
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Detection of AFor the detection of secreted
A
-(140), we used a specific sandwich enzyme-linked immunosorbent
assay employing monoclonal antibodies as described previously
(50).
Oxidative Stress-induced Cell DeathCell death was induced with hydrogen peroxide (freshly prepared solution; 250 µM; Sigma). In respective experiments, caspase inhibitors were added 3 h before exposure to H2O2 (250 µM, 24 h) at a final concentration of 10 µM: caspase 3 inhibitor (AC-DEVD-CMK), caspase 6 inhibitor (VEID-CHO), caspase 8 inhibitor (IETD-CHO), and caspase 9 inhibitor (LEHD-CHO). Only cell-permeable caspase inhibitors were used.
Measurement of Caspase ActivityPC12 cells were plated the
day before at a density of 5 x 106 cells/culture dish (10 cm
diameter). After treatment with H2O2, the cells were
harvested, and after a centrifugation step, the culture medium was aspirated.
The cell-containing pellet was washed with PBS and lysed in 100 µl of lysis
buffer (10 mM HEPES, 0.1% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 0.1 mM EDTA, 1 mM
dithiothreitol, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 0.1% CHAPS, pH
7.4). Lysates were centrifuged at 20,000 x g for 10 min at 4
°C, and the supernatant was used for caspase assay. The approximate
protein amount of the probes was confirmed by a Lowry protein test. Caspase
activity was measured by cleavage of colorimetric substrates (Ac-DEVD-pNA,
Ac-VEID-pNA, Ac-VDVAD-pNA, Ac-IETD-pNA, and
Ac-LEHD-pNA for caspase 3, caspase 6, caspase 2, caspase 8, and
caspase 9, respectively). The production of pNA was monitored over 30
min in a photometer ( = 405 nm). The caspase activity is expressed as
change in absorption units. One unit was defined as the amount of enzyme
required to cleave 1 pmol of NA per min of incubation per 5 x
106 cells.
Quantification of Apoptosis by Flow CytometryApoptosis was determined by propidium iodide (PI) staining and fluorescence-activated cell sorting analysis as described previously (12). Briefly, H2O2-treated PC12 cells were lysed in buffer (0.1% sodium citrate and 0.1% Triton X-100) containing 50 µg/ml propidium iodide. Samples were analyzed by flow cytometry (FACSCalibur) using Cell Quest software (Becton Dickinson). Cells with a lower DNA content showing less propidium iodide staining than G1 have been defined as apoptotic cells (sub- G1 peak).
Phospho-JNK Pathway Western Blot AnalysisPC12 cells were plated at a density of 5 x 106 cells/culture dish (10 cm diameter) and treated with H2O2. Cells were harvested and lysed in SDS sample buffer: Tris-HCl (62.5 mM, pH 6.8), 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.01% bromphenol blue. After sonicating and boiling, proteins were separated by electrophoresis on a 16% polyacrylamide gel. The separated proteins were transferred to a polyvinylidene difluoride membrane (ImmobilonTM-P; Millipore). These membranes were stained with Ponceau S red (reversible stain) to visualize the proteins. Nonspecific sites on the membranes were blocked with 5% fat-free milk in Tris-buffered saline, 0.1% Tween 20 (TBST). The phosphorylated JNK was visualized with the appropriate primary phosphoantibody, phospho-stress-activated protein kinase/JNK (Thr183/Tyr185), which detects stress-activated protein kinase/JNK only dually phosphorylated at Thr183/Tyr185. The phosphorylated c-Jun was detected by a phospho-c-Jun (Ser63) antibody. After thorough washing with TBST, membranes were covered with ECLTM detection reagents and quickly exposed to an autoradiography film. Membranes were routinely stripped for actin control.
Caspase Immunoblot AnalysisPC12 cells were plated at a density of 5 x 106 cells/culture dish (10 cm diameter) and treated with H2O2. After the indicated periods of time, the medium was aspirated, and cells were processed for Western blotting with rat-specific cleaved caspase 3, caspase 8, and caspase 9 antibodies. Blots were scanned (Umax Astra 4000 U), and the band intensity was determined after background subtraction using densitometric analysis.
Determination of Cytochrome c ReleaseThe amount of cytochrome c released from the mitochondrial intermembrane space into the cytosol was determined by digitonin permeabilization (51). Briefly, 5 x 106 cells were exposed to oxidative stress for different periods of time. After washing with ice-cold phosphate-buffered saline, cells were resuspended in permeabilization buffer containing 75 mM NaCl, 1 mM NaH2PO4, 8 mM Na2PO4, 250 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, additional protease inhibitors, and 0.05% digitonin. Following a centrifugation step at 800 x g at 4 °C for 10 min, the supernatant was separated from the pellet consisting of mitochondria and cellular debris. The supernatant containing cytoplasmic proteins was purified by centrifugation at 13,000 x g at 4 °C for 10 min. Equal amounts of protein (10 µg) were loaded on an 18% acrylamide gel and separated by SDS-PAGE. After immunoblotting with a monoclonal cytochrome c antibody, polyvinylidene difluoride membranes were stripped and reprobed either for COX4 or actin, ensuring equal protein loading and the absence of mitochondrial contamination.
Determination of Mitochondrial Membrane PotentialPC12 cells
were plated the day before at a density of 2 x 105 cells/well
in a 24-well plate. The cells were pretreated for 1 h with the JNK inhibitor
SP600125, and H2O2 was added for 6 h. The membrane
potential of the inner mitochondrial membrane was measured using the dye
rhodamine 123. The dye was added to the cell culture medium at a concentration
of 0.4 µM for 15 min. The cells were washed twice with Hanks'
balanced salt solution, and the fluorescence was determined with a
fluorescence reader (Victor Multilabel counter; PerkinElmer Life Sciences).
Transmembrane distribution of the dye depends on the mitochondrial membrane
potential (m).
Statistical AnalysisData are given as mean ± S.E. For statistical comparison, paired t test, Student's t test, or one-way ANOVA followed by Tukey's post hoc test or two-way ANOVA were used. p values less than 0.05 were considered statistically significant.
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RESULTS |
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APPsw Mutation Leads to an Enhanced Vulnerability to Oxidative Stress-induced ApoptosisWe induced apoptosis in transfected PC12 cells using the oxidative stressor hydrogen peroxide. Nuclear DNA fragmentation was quantitatively detected by propidium iodide staining and flow cytometry. Very importantly, clones bearing the APPsw mutation are sensitized to an exposure to oxidative stress for 24 h in a concentration-dependent manner with a maximum increase of apoptotic cells at a concentration of 500 µM H2O2 (12). Treatment with H2O2 led to a significantly enhanced maximum increase of apoptotic cells in APPsw-bearing PC12 cells (51.4 ± 5.3%) compared with APPwt-bearing cells (28.4 ± 4.7%) and vector control cells (21.3% ± 4.3%). Different clones with the same transfectant behave similarly in their sensitivity to H2O2-induced cell death (Fig. 1C).
Oxidative Stress Induces Activation of Caspase 2 in PC12
CellsOxidative stress in the human brain has been implicated as
one major cause of neuronal cell loss in AD patients. However, the exact
mechanism still remains unknown. A appears to bind redox-active metals
like zinc, copper, or iron with high affinity, resulting in production of
hydrogen peroxide and autoxidation of the metallopeptide complex
(52,
53). The signaling cascades
activated following the oxidative stress have not been widely studied.
Therefore, we studied the implication of caspase 2 by measuring the activity
and by Western blotting. Cleavage of the photometric substrate
Ac-VDVAD-p-nitroanilide by cytosolic protein extracts indicates the
presence of caspase 2 protease activity in the cultures after exposure to
hydrogen peroxide. The activity was already induced early in APPsw cells
within 2 h of induction (Fig.
2A). Caspase 2 activity was continuously elevated over
time compared with APPwt and vector-transfected cells. Maximum caspase 2
activity was measured after 2 h, with a 4-fold higher activity in APPsw cells
than in APPwt and vector PC12 cells, respectively
(Fig. 2B).
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The mechanism of activation of caspases occurs by a sequential cleavage of the zymogen to release the large and the small cleavage products. In accordance with our findings from the determination of activity, Western blotting confirmed cleavage of caspase 2 within 2 h of hydrogen peroxide induction (Fig. 2C). Again, the activation of caspase 2 was noticeably higher in APPsw cells compared with vector-transfected controls.
Involvement of Caspase 8 Apoptosis associated with the
Fas/tumor necrosis factor receptor family of death receptors requires caspase
8 activity and adaptor proteins such as FADD. The involvement of caspase 8 in
processing of APP during apoptosis by caspase 8 has been reported recently
(46). The blocking of neuronal
death by caspase 8 inhibitor IETD-fmk in A-induced cell death was also
demonstrated (54). Hence, we
examined the activation of caspase 8 in our transfected cell lines
overexpressing intracellular high levels of A
in order to ascertain the
involvement of this initiator caspase in oxidative stress. We utilized
Ac-IETD-pNA to measure caspase 8 activity in lysates of treated
cells. In parallel to the early activation of caspase 2, caspase 8 activity
increased about 3-fold after 2 h of stress induction in PC12 cells expressing
the Swedish double mutation and was significantly enhanced in a time-dependent
manner compared with vector transfected controls
(Fig. 3, A and
B). Activation of caspase 8 involves a two-step
proteolysis: the cleavage of the procaspase 8 to generate a 43-kDa fragment
and a 12-kDa fragment that is further processed to 10 kDa. The large fragment
is then cleaved to yield p26. Here, we used an antibody recognizing the 26-kDa
large fragment of caspase 8. Western blot analysis showed a notable expression
of active caspase 8 even in the untreated APPsw PC12 cells. Over the whole
time period, the protein expression in APPsw cells was continuously higher
compared with vector control (Fig.
3C).
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In addition, we analyzed the activities of caspase 1 and caspase 6 using AC-YVAD-pNA and AC-VEID-pNA. Interestingly, no changes in the activities of caspase 1 and caspase 6 were found in oxidative stress-induced cell death between mutant APP-bearing PC12 cells and controls (data not shown).
Increased Caspase 3 Activity of APPsw PC12 Cells in Response to Oxidative StressFurthermore, we prepared extracts from H2O2-treated PC12 cells and measured their ability to cleave the colorimetric substrate to Ac-DEVD-pNA, which is specifically cleaved by caspase 3. Cellular extracts from untreated control cultures showed very low Ac-DEVD-pNA cleavage in all cell lines (Fig. 4). Under these base-line conditions, no significant differences could be detected between vector-, APPwt-, and APPsw-transfected PC12 cells. After exposure to oxidative stress, caspase 3 activity increased gradually and reached a maximum activity after 6 h of incubation, demonstrating a delayed increase as compared with the initiator caspases 2 and 8. APPsw and APPwt PC12 cells showed a significantly enhanced caspase 3 activity compared with vector-transfected clones with the strongest increase in the APPsw cells (Fig. 4A). By Western blotting, we also found a significantly increased protein expression of the active caspase 3 in APPsw PC12 cells compared with control cells (Fig. 4B). The highest protein expression was evidenced after 6 h of H2O2 exposure by densitometric immunoblot analysis (Fig. 4C).
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Activation of Caspase 9 in Response to Oxidative Stress Mitochondria play a prominent role in the intrinsic apoptosis pathway. During mitochondrial dysfunction, several essential players of apoptosis, including procaspases, cytochrome c, and apoptosis-inducing factor, are released into the cytosol. The multimeric complex formation of cytochrome c, apoptotic protease-activating factor-1, and caspase 9 activates downstream caspases, leading to apoptotic cell death (55). Therefore, we examined whether oxidative stress in PC12 cells activates caspase 9 by using an polyclonal antibody specific for the inactive full-length procaspase 9 and for the cleaved fragments of the protein. We detected a time-dependent increase in cleaved fragments of caspase 9 after treatment of the cells with hydrogen peroxide (Fig. 5A). APPsw PC12 cells showed an elevated expression of the active cleaved fragments of caspase 9 compared with vector-transfected cells. A time-dependent cytochrome c increase in the cytosol was also detected by Western blot using a specific antibody (Fig. 5D). The highest cytochrome c amount in the cytosolic fraction was detected after a 4-h oxidative stress induction. In addition, caspase 9 activities were measured colorimetrically using Ac-LEHD-pNA as substrate in lysates of hydrogen peroxide-treated cells (Fig. 5B). We ascertained an early induced activation of caspase 9 after 2 h, whereas the activity was increased in the APP-transfected cells compared with vector-transfected controls but was not different between APPsw and APPwt cells (Fig. 5C).
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Enhanced Activation of JNK Pathway in APPsw PC12 CellsThe
mobilization of the stress-activated protein kinase (JNK) plays an important
role in apoptosis induced by several types of environmental stress, like UV,
-radiation, inflammatory cytokines, and DNA damage
(5658).
In oxidative stress-induced cell death, the activation of mitochondrial
apoptosis machinery by JNK in adult cardiac myocytes has been observed
(37). Hence, we addressed the
question whether oxidative stress-mediated activation of the JNK pathway is
affected by overexpression of APP. We performed a Western blotting of
immunocomplexes to detect activation of JNK and c-Jun.
H2O2 treatment of APPsw PC12 cells induced JNK
phosphorylation within 2 h (Fig.
6A). The highest expression was detected after 4 h with
an elevated expression in APPsw cells compared with APPwt cells and vector
control. When activated, JNK translocates to the nucleus to regulate
transcription through its effects on c-Jun and other transcription factors. As
for JNK, we observed a strong impact of the Swedish APP transfection on the
activation of c-Jun (Fig.
6B). In our experiments, the kinetics of activation of
c-Jun correlated with the phosphorylation of JNK with the highest detection of
phosphorylated c-Jun after 6 h of H2O2 treatment. The
expression of phosphorylated c-Jun was markedly elevated in APPsw compared
with treated APPwt or vector-transfected controls after 4 and 6 h of hydrogen
peroxide treatment.
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Prevention of Apoptosis by Caspase InhibitorsDue to the enhanced activation of several caspases in response to oxidative stress, we examined the potential against several caspase inhibitors in protecting PC12 cells of oxidative stress-induced apoptosis. Caspase inhibitors were added to cultures of PC12 cells in a time- and concentration-dependent manner (data not shown). Best protection was provided by adding caspase inhibitors at a concentration of 10 µM 3 h prior to H2O2 exposure. As shown in Fig. 7, only the inhibitor of the effector caspase 3, AC-DEVD-CMK, was able to rescue APPsw cells from apoptosis below the level of that from vector-transfected control cells. The pretreatment of the APPsw cells with inhibitors of initiator caspase 8 and caspase 9 revealed similar levels than did vector cells.
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Inhibiton of JNK by SP600125As detected by Western blotting, we could show that the transfection of the Swedish FAD mutation in PC 12 cells leads to an enhanced activation of the JNK pathway under oxidative stress. Therefore, we investigated the effect of SP600125, a selective anthrapyrazolone inhibitor of JNK (59), in our cell model. We measured the caspase activities of different caspases and the mitochondrial membrane potential after pretreatment with JNK inhibitor to attribute the JNK involvement in oxidative stress-induced apoptosis. We found a highly significant reduction of caspase 9 activity (two-way ANOVA, p < 0.001) and caspase 3 activity (two-way ANOVA, p = 0.0384) in cell lysates of APPsw cells pretreated with 200 nM JNK inhibitor, whereas the activities of caspase 2 and caspase 8 were not significantly altered (Fig. 8). Corresponding to the reduction of caspase 9 activity, we were able to detect a protection against a decrease in the mitochondrial membrane potential by SP600125 (Fig. 9), indicating an involvement of JNK upstream of mitochondria in oxidative stress signaling. Whereas the treatment with H2O2 (250 µM) led to a significant increase of apoptotic cells in APPsw-bearing PC12 cells (42.21 ± 3.71%) compared with vector cell line (30.87 ± 5.97%), APPsw cells pretreated with the JNK inhibitor (33.77 ± 4.06%) exhibited similar apoptotic cell levels as the corresponding vector cells (Fig. 7).
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DISCUSSION |
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Therefore, studies of A-induced neurotoxic effects under more likely
physiological conditions mimicking the situation in AD brains are needed
(60). Moreover, A
is
produced intracellularly and can accumulate within cells
(21,
61,
62) before its deposition in
senile plaques. Intracellular accumulation of A
might impair cellular
functions and may represent the primary event within the neurotoxic A
cascade. In order to mimic the situation in vivo, we decided to
transfect PC12 cells with human APP and the FAD-related Swedish APP mutation.
APPsw and APPwt PC12 cells showed a moderate expression of human APP, leading
to low levels of A
-(140) production (APPsw, 90 pg/ml; APPwt, 20
pg/ml) close to the situation in vivo. These two cell lines with
different production levels of A
may additionally allow us to study
dose-dependent effects of A
. Expression of APPsw rendered PC12 cells
more vulnerable to the induction of cell death after exposure to oxidative
stress. It seems likely that increased production of A
at physiological
levels primes APPsw cells to undergo cell death only after additional stress,
a scenario that has been suggested to occur in AD brain. Moreover, we were
able to show that caspase 3 activity was significantly enhanced in APPsw
compared with APPwt and vector-transfected cells. Interestingly, APPwt also
showed an increased activity of caspase 3 compared with empty vector control
cells, although to a lesser extent than APPsw cells. Our data suggest that
already very low A
levels in APPwt cells are probably sufficient to
prime cells to undergo apoptosis. This is confirmed by our findings that
caspase 9 activity is increased and at the same time mitochondrial membrane
potential is lowered in APPwt as well as in APPsw cells compared with
vector-transfected control cells after the induction of oxidative stress. In
addition, we examined the implication of two other initiator caspases, which
has been described primarily in the extrinsic apoptotic pathway. We
demonstrated that caspase 8, involved in death receptor signaling, is
activated in oxidative stress signaling in PC12 cells. Importantly, this
activation is markedly elevated in APPsw cells compared with APPwt cells.
Whether this increase is due to effects of extracellularly secreted A
on
cell death receptors or is mediated by intracellularly accumulated A
is
not clear and still under investigation. In parallel, we observed in PC12
cells an activation of caspase 2 in oxidative stress signaling. In APPsw
cells, the maximal activity was 4-fold higher compared with APPwt and vector
PC12 cells, respectively. This finding is of particular interest, since recent
data describe caspase 2 to be localized in several intracellular compartments,
including the mitochondria, Golgi, cytosol, and nucleus
(6365).
Cytotoxic stress causes activation of caspase 2, which is required for the
permeabilization of mitochondria
(66) and induces the release
of cytochrome C (64,
67,
68). It is assumable that the
enhanced A
production in the APPsw-transfected cells occurs through an
increased expression and activity of beta-site APP-cleaving enzyme
(26). The elevated
intracellular levels of A
in the mutant cells may therefore lead to an
enhanced activation of caspase 2, which in turn implements the activation of
the intrinsic apoptotic pathway by permeabilizing mitochondria
(69). As a consequence,
cytochrome c is released to the cytosol, resulting in an activation
of the apoptotic protease-activating factor-1/caspase 9 apoptosome complex.
Simultaneously, other effects of caspase 2 need to be considered, since we
determined no differences in cytochrome C release and caspase 9 activation
between APPsw and APPwt cells. Recent data indicate that A
induces the
release of other proapoptotic factors from the mitochondria (e.g.
Diablo/Smac (second mitochondria-derived activator of caspase))
(70). Thus, Smac or other
unknown factors may be involved in our cell model. Since caspase 2 activation
occurs early in our cell model and neurons from caspase 2 null mice are
resistant to
-amyloid-induced death
(47), this caspase seems to
play a critical role.
Given the important role that mitogen-activated protein kinase pathways
play in cellular stress signaling, we investigated the relationship between
activated JNK, apoptosis, and the Swedish APP mutation. We demonstrated that
oxidative stress leads to an activation of JNK. JNK phosphorylates specific
sites in the N-terminal region of c-Jun, thereby enhancing its transcriptional
activity. Very importantly, we were able to demonstrate that in PC12 cells
bearing mutant APPsw, the activation of JNK and c-Jun was significantly
enhanced compared with APPwt cells. At the same time, activation of JNK and
c-Jun is increased in APPwt compared with vector cells. This suggests that the
different A levels induce a dose-dependent activation of JNK pathway.
Interestingly, we could observe a reduction of caspase 9 activity using the
selective JNK inhibitor SP600125, whereas activities of caspase 2 and caspase
8 were unaltered. Furthermore, the inhibitor compensated the mitochondrial
abnormality when oxidative stress was induced. Due to the fact that members of
the antiapoptotic Bcl-2 family proteins are inactivated through
phosphorylation by JNK (71,
72), our results support
evidence that JNK alters mitochondrial function. Recently, it was reported by
different groups that JNK-interacting proteins JIP1b and JIP2 bind the
cytoplasmic domain of APP
(7376).
JIP family proteins are scaffold proteins that organize specific members of
the JNK/mitogen-activated protein kinase cascade to facilitate signaling. It
was proposed that these scaffold proteins could serve as cargo for the
microtubule motor kinesin to mediate the transportation of several
transmembrane proteins (77),
in which APP is supposed to be a cargo receptor
(78). Our data indicate that
the altered processing of APPsw might play a crucial role in activation of the
stress kinase pathway. Whether this occurs by a redistribution of these
scaffold proteins has to be clarified in the future.
In addition, we investigated the protective effects of caspase inhibitors as well as JNK inhibitor in preventing oxidative stress-mediated cell death. Only the caspase 3 inhibitor was able to reduce oxidative stress-induced apoptosis significantly in APPsw cells below the level of control cells. The inhibition of caspase 8 or caspase 9 was less effective in preventing apoptosis. The lack of efficacy of the initial caspase inhibitors to abolish completely apoptotic cell death could be attributed to the compensatory pathway. Although the treatment of PC12 cells with hydrogen peroxide leads to activation of these initiator caspases, their inhibition is not enough to avoid apoptosis, because different caspase pathways co-exist (Fig. 10). Such a compensation of caspase pathway was ascertained in sympathetic neurons from caspase 2 null animals. A lack in caspase 2 expression was compensated by an increased expression of both mRNA and protein for caspase 9 and DIABLO/Smac in trophic factor deprivation-mediated death (79).
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In summary, we propose a hypothetical sequence of events linking FAD,
A production, JNK activation, and mitochondrial dysfunction with caspase
pathway and neuronal loss for our cell model
(Fig. 10). The brain has a
high metabolic rate and is exposed to gradually rising levels of oxidative
stress during life. In Swedish FAD patients, the levels of oxidative stress
are increased in the temporal inferior cortex
(28). Our study using a cell
model mimicking the in vivo situation in AD brains indicates that
probably both increased A
production and the gradual rise of oxidative
stress throughout life lead to an increased vulnerability to apoptotic cell
death in neurons from FAD patients.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Biocenter of the Johann Wolfgang Goethe-University, Bldg. N260, Marie-Curie-Strasse 9, D-60439 Frankfurt am Main, Germany. Tel.: 49-69-79829380; Fax: 49-69-79829374; E-mail: A.Eckert{at}em.uni-frankfurt.de.
1 The abbreviations used are: A, amyloid-
-protein; APP, amyloid
precursor protein; AD, Alzheimer's disease; FAD, familial Alzheimer's disease;
APPsw, Swedish double mutation form of APP; APPwt, wild-type APP; JNK, c-Jun
N-terminal kinase; pNA, para-nitroaniline; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PI, propidium
iodide; ANOVA, analysis of variance; CHO, aldehyde.
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REFERENCES |
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