THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 279, No. 48, Issue of November 26, pp. 50310 –50320, 2004
Printed in U.S.A.
Amyloid ␤-induced Changes in Nitric Oxide Production and
Mitochondrial Activity Lead to Apoptosis*
Received for publication, May 19, 2004, and in revised form, September 8, 2004
Published, JBC Papers in Press, September 14, 2004, DOI 10.1074/jbc.M405600200
Uta Keil‡, Astrid Bonert‡, Celio A. Marques‡, Isabel Scherping‡, Jörg Weyermann§,
Joanna B. Strosznajder ¶, Franz Müller-Spahn储, Christian Haass**, Christian Czech‡‡§§,
Laurent Pradier§§, Walter E. Müller‡, and Anne Eckert‡储¶¶
From the ‡Departments of Pharmacology and §Pharmaceutical Technology, Biocenter, University of Frankfurt, 60439
Frankfurt, Germany, ¶Department of Cellular Signaling, Medical Research Center, Polish Academy of Sciences, 02-106
Warsaw, Poland, 储Neurobiology Research Laboratory, Psychiatric University Clinic, 4025 Basel, Switzerland,
**Department of Biochemistry, Adolf Butenandt Institute, 80336 Munich, Germany, and §§Aventis Pharma,
Research & Development, F-94403 Vitry-sur-Seine, France
Alzheimer’s disease (AD)1 is the most common neurodegenerative disorder (1) marked by progressive loss of memory and
* This work was supported by grants from the Alzheimer Forschung
Initiative (to A. E.), German Polish Grant PBZ-MIN-0011P05116, and
from the Deutsche Forschungsgemeinschaft (to C. A. M.). The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡‡ Present address: Hoffman-La Roche AG, CNS Research, CH-4070
Basel, Switzerland.
¶¶ 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.: 49-69-79829377; Fax:
49-69-79829374; E-mail: [email protected].
1
The abbreviations used are: AD, Alzheimer’s disease; A␤, amyloid ␤
peptide; APP, amyloid precursor protein; FAD, familial Alzheimer’s
disease; APPsw, Swedish double mutation form of APP; APPwt, wild-
cognitive ability. The pathology of AD is characterized by the
presence of amyloid plaques (2) and intracellular neurofibrillary tangles and pronounced cell death. The amyloid plaque is
composed of amyloid ␤ (A␤) peptide (3), which is derived from
the amyloid precursor protein (APP) through an initial ␤-secretase cleavage followed by an intramembraneous cut of ␥-secretase (4, 5). Autosomal dominant forms are caused by mutations
in APP, presenilin 1, and presenilin 2, mainly associated with
the early onset of AD (6). The Swedish double mutation in the
APP gene (K670M/N671L) results in a 6 – 8-fold increased A␤
production compared with human wild type APP cells (APPwt)
(7, 8). We have previously shown that the APPsw mutation
enhances the vulnerability to secondary insults, e.g. oxidative
stress, finally leading to apoptotic cell death through the activation of the c-Jun N-terminal kinase and caspases 3 and 9 (9).
The latter observation provided evidence that mitochondriamediated apoptosis might play an important role in these
processes.
Intrinsic apoptotic pathway via mitochondria is regulated by
members of the Bcl-2 family (10). They are mainly localized in
the outer mitochondrial membrane. Bcl-2 and Bcl-xL inhibit
apoptosis, whereas other members such as Bax, Bak, Bid, Bik,
and Bim are proapoptotic (11). Their most frequently reported
mode of action is the regulation of cytochrome c release from
the intermembrane space (12). Once released from the mitochondria, cytochrome c interacts with Apaf 1 and procaspase 9
to activate caspase 3, finally leading to cell death. Smac (second
mitochondria-derived activator of caspase) is another mitochondrial intermembrane protein that is released in the cytosol
during apoptosis (13). Smac interacts with several inhibitors of
apoptosis, thereby relieving the inhibitory effect of inhibitors of
apoptosis on caspases (14). As an early event in the apoptotic
pathway, typically, a rapid reduction of the mitochondrial
membrane potential (⌿m) takes place (15), which reflects a
block of the respiratory chain finally leading to the reduction of
ATP levels.
Some previous findings suggest that neurotoxicity of A␤ (16,
17) seems to be mediated via oxidative stress probably leading
to mitochondrial damage, e.g. A␤ inhibited cytochrome c oxidase (COX) activity in isolated brain mitochondria (18, 19), and
type APP; NO, nitric oxide; NOS, nitric-oxide synthase; ELISA, enzyme-linked immunosorbent assay; HEK, human embryonic kidney; tg,
transgenic; COX, cytochrome c oxidase; ROS, reactive oxygen species;
⌿m, mitochondrial membrane potential; Smac, second mitochondriaderived activator of caspase; AIF, apoptosis-inducing factor; Tricine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TMRE, tetramethylrhodamineethyl ester; ANOVA, analysis of variance; DAPT, N-[N(3,5-trifluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester.
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This paper is available on line at http://www.jbc.org
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Increasing evidence suggests an important role of mitochondrial dysfunction in the pathogenesis of Alzheimer’s disease. Thus, we investigated the effects of acute
and chronic exposure to increasing concentrations of
amyloid ␤ (A␤) on mitochondrial function and nitric oxide
(NO) production in vitro and in vivo. Our data demonstrate that PC12 cells and human embryonic kidney cells
bearing the Swedish double mutation in the amyloid precursor protein gene (APPsw), exhibiting substantial A␤
levels, have increased NO levels and reduced ATP levels.
The inhibition of intracellular A␤ production by a functional ␥-secretase inhibitor normalizes NO and ATP levels, indicating a direct involvement of A␤ in these processes. Extracellular treatment of PC12 cells with
comparable A ␤ concentrations only leads to weak
changes, demonstrating the important role of intracellular A␤. In 3-month-old APP transgenic (tg) mice, which
exhibit no plaques but already detectable A␤ levels in the
brain, reduced ATP levels can also be observed showing
the in vivo relevance of our findings. Moreover, we could
demonstrate that APP is present in the mitochondria of
APPsw PC12 cells. This presence might be directly involved in the impairment of cytochrome c oxidase activity
and depletion of ATP levels in APPsw PC12 cells. In addition, APPsw human embryonic kidney cells, which produce 20-fold increased A␤ levels compared with APPsw
PC12 cells, and APP tg mice already show a significantly
decreased mitochondrial membrane potential under basal conditions. We suggest a hypothetical sequence of
pathogenic steps linking mutant APP expression and
amyloid production with enhanced NO production and
mitochondrial dysfunction finally leading to cell death.
Amyloid ␤, Nitric Oxide, and Mitochondria
EXPERIMENTAL PROCEDURES
Materials—Rhodamine 123 and tetramethylrhodamineethylester
(TMRE) were purchased from Molecular Probes. 4,5-Diaminofluorescein diacetate was obtained from Calbiochem. ViaLight HT kit was
purchased from Cambrex. Cytochrome c oxidase assay kit, hydrogen
peroxide, rotenone, thenoyltrifluoroacetone, antimycin, sodium azide
(NaN3), and oligomycin were obtained from Sigma. L-NAME was purchased from Cayman. N␻-propyl-L-arginine and 1400W were purchased
from Biotrend. A␤1– 42 was supplied by Bachem. DAPT was obtained
from Merck Biosciences.
A␤1– 42 was dissolved in Tris-buffered saline (pH 7.4) at a concentration of 1 mM and stored at ⫺20 °C. The stock solution was diluted in
Tris-buffered saline to the desired concentrations and incubated at
37 °C for 24 h to have aged preparations of A␤1– 42.
Cell Culture and Transfection—PC12 cells and HEK cells were transfected with DNA constructs harboring human mutant APP (APPsw,
K670M/N671L) gene, the APPwt gene, inserted downstream of a cytomegalovirus promoter using the FUGENE 6 technique (Roche Diagnostics) (37). The transfected cells APPwt PC12, APPsw PC12, and control
PC12 were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal calf serum and 5% heat-inactivated horse serum, 50 units/ml penicillin, 50 ␮g/ml streptomycin, and
400 ␮g/ml G418 at 37 °C in a humidified incubator containing 5% CO2.
The transfected cell lines APPwt HEK, APPsw HEK, and control
HEK were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal calf serum, 50 units/ml penicillin, 50 ␮g/ml streptomycin, and 400 ␮g/ml G418 at 37 °C in a humidified incubator containing 5% CO2. The A␤ levels of APP-transfected
PC12 cells and HEK cells are shown in Table I.
Detection of A␤ levels—For the detection of secreted A␤1– 40, a specific
sandwich enzyme-linked immunosorbent assay (ELISA) employing
monoclonal antibodies was used. The ELISA was performed according
to the instructions given in the Abeta-ELISA kit by BIOSOURCE. The
assay principle is that of a standard sandwich ELISA, which utilizes a
monoclonal mouse anti-human Abeta1–16 capture antibody, a cleavage
site-specific rabbit anti-human Abeta1– 40 C-terminal detection antibody, and anti-rabbit IgG peroxidase-conjugated secondary antibody.
Transgenic Animal Brain Tissue—Female C57BL/6 mice bearing the
human Swedish and London mutations in the 751 amino acid form of
human amyloid precursor protein (tg APP) under the control of a
murine Thy-1 promoter at an age of 3 months and non-transgenic
littermate animals were used for the experiments (38). APP tg mice
exhibited the onset of A␤ plaques at an age of 6 months, but intracellular A␤ load was already detectable at the age of 3 months (39).
Mice were sacrificed by decapitation, and brains were quickly dissected on ice (method modified after Stoll et al. (40)). After removing the
cerebellum, the tissue was minced into 2 ml of medium I (138 NaCl, 5.4
KCl, 0.17 Na2HPO4, 0.22 K2PO4, 5.5 glucose, and 58.4 sucrose, all in
mmol/liter, pH 7.35) with a scalpel and further dissociated by trituration through a nylon mesh (pore diameter 1 mm) with a Pasteur pipette.
The resulting suspension was filtered by gravity through a fresh nylon
mesh with a pore diameter of 102 ␮M, and the dissociated cell aggregates were washed twice with medium II (110 NaCl, 5.3 KCl, 1.8
CaCl2䡠H2O, 1 MgCl2䡠6 H2O, 25 glucose, 70 sucrose, and 20 HEPES, all
in mmol/l, pH 7.4) by centrifugation (400 ⫻ g for 3 min at 4 °C). 100 ␮l
of the suspension were used for protein determination. After centrifugation, cells were resuspended in 6 ml of Dulbecco’s modified Eagle’s
medium and 500 ␮l/well were distributed on a 24-well plate for the
measurement of mitochondrial membrane potential. For the measurement of ATP levels, 100 ␮l/well were distributed on a white 96-well
plate. The preparations of APP tg mice and non-transgenic littermate
mice (overcross design) were made under the same conditions and
in parallel.
Quantification of Apoptosis by Flow Cytometry—Apoptosis was determined by propidium iodide staining and fluorescence-activated cell
sorter analysis as described previously (37). PC12 cells and HEK cells
were lysed in buffer (0.1% sodium citrate and 0.1% Triton X-100)
containing 50 ␮g/ml propidium iodide. Samples were analyzed by flow
cytometry (FACSCalibur) using Cell Quest software (BD Biosciences).
Cells with a lower DNA content showing less propidium iodide staining
than G1 have been defined as apoptotic cells (sub-G1 peak).
Determination of Intracellular Nitric Oxide Levels—PC12 cells and
HEK cells were plated the day before at a density of 2 ⫻ 105 cells/well
in a 24-well plate. To measure the NO levels, the fluorescence dye
4,5-diaminofluorescein diacetate was used in a concentration of 10 ␮M
for 30 min (41). The cells were washed twice with Hanks’ balanced salt
solution, and the fluorescence was determined with a fluorescence
reader (Victor® multilabel counter) at 490/535 nm.
NO levels were also determined after a 48-h incubation in the absence or in the presence of the NO synthase inhibitors 20 mM L-NAME,
1 mM N␻-propyl-L-arginine, and 1 mM 1400W.
Determination of the Mitochondrial Membrane Potential (␺m)—PC12
cells and HEK cells were plated the day before at a density of 2 ⫻ 105
cells/well in a 24-well plate. PC12 cells were incubated with H2O2 (0.5
mM) for different periods of time. The mitochondrial membrane potential was measured using the fluorescence dye Rhodamine 123 (42).
Transmembrane distribution of the dye depends on the mitochondrial
membrane potential. The dye was added to the cell culture medium in
a concentration of 0.4 ␮M 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) at 490/535 nm.
The mitochondrial membrane potential of dissociated neurons was
also measured with Rhodamine 123 in a concentration of 0.4 ␮M for 15
min. For detailed information regarding the preparation, see “Transgenic Animal Brain Tissue.”
To test acute and fast changes in ␺m, the fluorescence dye TMRE (43)
was used in a concentration of 0.4 ␮M for 15 min. TMRE exhibits a
characteristic increase in fluorescence at 490/590 nm after challenging
mitochondria with membrane potential-decreasing drugs (44). The mitochondrial membrane potential was recorded, and then the complex
inhibitors (2 ␮M rotenone, 10 ␮M thenoyltrifluoroacetone, 2 ␮M antimycin, 10 ␮M oligomycin, and 10 mM sodium azide) were added.
Determination of ATP Levels with a Bioluminescence Assay (ViaLight
HT)—PC12 cells and HEK cells were plated the day before at a density
of 2 ⫻ 104 cells/well in a white 96-well plate. PC12 cells were incubated
for different periods of time with H2O2 (0.1 mM).
The ATP levels of dissociated neurons were also measured with the
bioluminescence assay. For detailed information regarding this preparation, see “Transgenic Animal Brain Tissue.”
The kit is based upon the bioluminescent measurement of ATP (45).
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in neuronal cultures, A␤ caused a loss of activity of all mitochondrial respiratory chain complexes (20 –23). Moreover, activity changes in mitochondrial enzymes including pyruvate
dehydrogenase and ␣-ketoglutarate dehydrogenase have been
described in AD brain (24). In addition, patients with AD
showed impaired cytochrome c oxidase activity in the central
nervous system and even in other tissues including platelets
(19, 25–27). Interestingly, p0 cells repopulated with mitochondria from AD patients also showed a reduced cytochrome c
oxidase activity (28). Thus, the defects in mitochondrial energy
metabolism that lead to increased production of reactive oxygen species (ROS) seem to underlie the pathology of AD (29).
Mitochondria represent not only the major source of ROS but
also the main target for ROS damage. Nitric oxide (NO) and its
derivative (reactive nitrogen species), also belonging to the
group of ROS, are known to inhibit the mitochondrial respiration (30). Hereby, NO itself causes a selective reversible inhibition of cytochrome c oxidase (31), whereas RNS inactivate
multiple respiratory chain complexes and ATP synthase (32).
In accordance, both oxidative and nitrosative stress seem to
represent early events in the pathogenesis of AD (33–36).
On the basis of these findings, we set out to investigate the
precise mechanisms underlying the action of A␤ and/or expression of mutant APP on mitochondrial function in multiple
experimental designs mimicking different in vivo situations
that are discussed to occur in AD patients: 1) in cell lines
overexpressing different levels of A␤ (APPsw PC12 cells exhibiting low physiological concentrations of A␤ within picomolar
range and APPsw HEK cells expressing A␤ levels within low
nanomolar range) to study dose-dependent effects of A␤ in an
in vitro setting characterized by chronic A␤ production due to
increased APP processing; 2) after extracellular A␤ treatment
to distinguish chronic from acute and/or extracellular actions
from effects of intracellular A␤ or APP-processing products on
mitochondria; and 3) in a secondary insult model to examine
the additional impact of oxidative stress. Finally, we checked
the in vivo relevance of our in vitro findings by studying mitochondrial function in brain cells from APP transgenic (tg) mice.
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Amyloid ␤, Nitric Oxide, and Mitochondria
50312
TABLE I
A␤ secretion of PC12 cells and HEK cells in pg/ml medium unter basal conditions and after incubation with DAPT
One-way ANOVA, post hoc Tukeys multiple comparison test. Values are means ⫾ S.E. from 3– 4 experiments.
PC12 cells
HEK cells
a
b
Co
APPwt
APPsw
3.67 ⫾ 1.52
35.47 ⫾ 1.16
17.33 ⫾ 3.18
259.1 ⫾ 7.91a
97.33 ⫾ 9.56
2598 ⫾ 26.29a
a
APPsw ⫹ DAPT (24 h 1 ␮M)
APPsw ⫹ DAPT (48 h 1 ␮M)
19.81 ⫾ 0.40
73.69 ⫾ 0.65b
19.44 ⫾ 0.41b
15.77 ⫾ 0.15b
b
p ⬍ 0.001 versus control PC12 and HEK cells.
p ⬍ 0.001 versus untreated APPsw cells.
RESULTS
APP Expression and A␤ Levels of APP-transfected PC12 Cells
and HEK Cells—Increasing knowledge was obtained that A␤ is
produced intracellularly (48) and that intraneuronal accumulation of A␤ precedes plaque formation in APP tg mice bearing
the Swedish double mutation (49, 50). Thus, intracellular accumulation of A␤ might be a primary step in the neurotoxicity
cascade of A␤ in vivo and in vitro. APP containing the Swedish
mutation resulted in an overall increase of A␤ including both
A␤1– 40 and A␤1– 42.
In our cell model, the APPsw mutation resulted in a markedly increased A␤ secretion of PC12 cells (0.20 nmol/liter) as
well as in HEK cells (5.42 nmol/liter) compared with APPwt
PC12 cells and APPwt HEK cells. APPsw PC12 cells secreted
low A␤ levels, reflecting the physiological situation in vivo
during cellular metabolism (7), whereas human APP was
equally expressed in APPwt and APPsw PC12 cells and was
increased 2-fold compared with the endogenous APP expression of control PC12 cells (37). Of note, HEK cells bearing the
APPsw mutation exhibited an ⬃20-fold increased A␤ secretion
compared with APPsw-bearing PC12 cells (Table I), a scenario
that might occur in familial AD and that could explain the more
drastic effects in some assay designs. Furthermore, this might
indicate that, in APPsw HEK cells, the secretion pathway is
more pronounced than in PC12 cells. The endogenous human
APP expression of control HEK cells was increased 2-fold compared with APPsw PC12 cells. Moreover, the APP expression of
APPwt and APPsw HEK cells was increased 3-fold compared
with control HEK cells and 6-fold compared with APPsw PC12
cells. Using the different cell lines allowed us to study the
dose-dependent effects of A␤ on those stably transfected cells
with APPwt or APPsw, both representing a model of chronic
A␤ stress.
Basal Apoptosis Was Increased in APPsw HEK Cells but Not
in APPsw PC12 Cells—Apoptotic cell death plays a very important role in the pathogenesis of AD (51). Thus, we investigated
basal apoptosis using propidium iodide staining. In APPsw
PC12 cells, we observed no increased basal apoptosis (Fig. 1A).
Thus, APPsw PC12 cells were able to compensate for the consequences of the increased A␤ levels. However, APPsw HEK
cells, which have high A␤ levels, showed significantly increased
levels of apoptotic cells under base-line conditions (Fig. 1B).
Additionally, the treatment of PC12 cells with extracellular
A␤1– 42 at concentrations of 10 nM and higher resulted also in
increased apoptotic cell death (Fig. 1C). The effect on the induction of apoptosis was less pronounced after extracellular A␤
treatment than in APPsw HEK cells. Even the A␤1– 42 peptide
was used, which is more toxic than the A␤1– 40 peptide.
Basal NO Levels Were Increased in APPsw Cells—Excessive
generation of nitric oxide has been implicated in the pathogenesis of AD (52). Thus, we measured intracellular NO levels
using the fluorescence dye 4,5-diaminofluorescein diacetate in
APP-transfected PC12 cells and HEK cells. Our data demonstrated that NO levels are significantly enhanced in the following order: control cells ⬍ APPwt ⬍ APPsw (Fig. 1, E and F) in
both cell lines at a similar level. To determine the contribution
of extracellular A␤ on NO levels, we treated PC12 cells extra-
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The bioluminescent method utilizes an enzyme, luciferase, which catalyzes the formation of light from ATP and luciferin. The emitted light is
linearly related to the ATP concentration and is measured using a
luminometer.
Confocal Microscopy—The amount of mitochondria was measured
using the cell-permanent mitochondrion-selective dye Mitotracker Red.
This probe can accumulate in active mitochondria and then react with
accessible thiol groups of proteins and peptides to form fluorescent
aldehydic-fixable conjugates. PC12 cells were plated the day before at a
density of 2 ⫻ 105 cells/chamber slide. Cells were incubated for 30 min
at 37 °C with Mitotracker Red or for 15 min with Rhodamine 123. Cells
were washed twice with phosphate-buffered saline, fixed with 2%
paraformaldehyde for 15 min, and then washed twice with phosphatebuffered saline. The samples were embedded in Mowiol and analyzed
using a laser-scanning confocal microscope.
Isolation of Cytosolic and Mitochondrial Fractions—Cytosolic and
mitochondrial fractions were isolated by digitonin permeabilization
(46). 5 ⫻ 106 cells were washed with ice-cold phosphate-buffered saline,
and cells were resuspended for 15 min on ice in permeabilization buffer
containing 75 mM NaCl, 1 mM NaH2PO4, 250 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, additional protease inhibitors, and 0.05%
digitonin. Following a centrifugation step at 800 ⫻ g at 4 °C for 10 min,
the supernatant was separated from the pellet consisting of cellular
debris. The crude mitochondrial pellet was collected by centrifugation
at 13,000 ⫻ g at 4 °C for 10 min. The supernatant containing cytoplasmic proteins was stored at ⫺20 °C for further investigation. The pellet
consisting of mitochondria was dissolved in phosphate-buffered saline
for the cytochrome c oxidase assay and resuspended in 0.1% Triton
X-100 and mechanically lysed for the Western blot. The total protein
content was determined by the method of Lowry (Bio-Rad).
Determination of Cytochrome c Oxidase Activity in Isolated Mitochondria with Cytochrome c Oxidase Assay Kit (47)—The colorimetric assay
is based on the observation of the decrease in absorbance at 550 nm of
ferrocytochrome c caused by its oxidation to ferricytochrome c by cytochrome c oxidase. The cytochrome c oxidase assay was performed according to the instructions given in the kit.
Western Blot—After determination of the total protein content by the
method of Lowry, the cytosolic and mitochondrial fraction was mixed
with 4⫻ Laemmli sample buffer and denatured for 10 min at 95 °C. An
equal amount (10 –20 ␮g) of protein was loaded per lane on acrylamide
gels and examined by SDS-PAGE. Dependent on the protein to be
detected, we performed glycine or Tricine Western blots with acrylamide amounts of 10 –18% at 90 V for 2–3 h. For proteins with a molecular
mass ⬍30 kDa, Tricine Western blots were more convenient. The proteins were transferred onto polyvinylidene difluoride membranes (Amersham Biosciences) at 25 V for 90 min. After this procedure, membranes were saturated with 5% nonfat dry milk in Tris-buffered saline
with Tween 20 for 1 h after antibody exposure. The polyvinylidene
difluoride membranes then were exposed to the following antibodies
overnight: rabbit anti-Bcl-xL (Cell Signaling); rabbit anti-Bax (Cell Signaling); rabbit anti-apoptosis-inducing factor (AIF) (Chemicon); rabbit
anti-Smac (Chemicon); rabbit anti-cytochrome c (BD Biosciences),
mouse anti-COX4 (BD Biosciences); goat anti-actin (Santa Cruz Biotechnology); rabbit anti-calreticulin (Chemicon); mouse anti-Na⫹/K⫹ATPase (Chemicon); rabbit anti-nitrotyrosine (Merck Biosciences); and
mouse anti-human APP W02 (Abeta; W02 antibody is directed against
amino acids 4 –10 of the human A␤ sequence, thereby detecting the
mature and immature APP695). After treatment for 1 h with the corresponding horseradish peroxidase-coupled secondary antibodies
(Merck Biosciences), the protein bands were detected by ECL reagent
(Amersham Biosciences). After detection, the membranes were treated
with stripping buffer (100 mM glycine, pH 2.5) for 2 h after reprobing
with different antibodies.
Statistical Analysis—The data are given as the mean ⫾ S.E. For
statistical comparison, Student’s t test and one-way ANOVA followed by
Tukey’s post hoc test, repeated measures ANOVA, or two-way ANOVA
were used. p values ⬍ 0.05 were considered statistically significant.
Amyloid ␤, Nitric Oxide, and Mitochondria
50313
cellularly with A␤1– 42 (Fig. 1G). Interestingly, NO levels were
also enhanced after exposure to A␤1– 42 with a maximal effect
already at 1 nM but to a lesser extent than in APPsw PC12 cells.
Thus, the extracellularly applied A␤ cannot completely mimic
the physiological situation of APPsw transfection.
NO Synthase (NOS) Activity Was Enhanced in APPsw
Cells—NO was synthesized from L-arginine by three isoforms
of NOS, two of which (endothelial NOS and neuronal NOS)
were constitutively expressed, whereas the third (inducible
NOS) was induced during inflammation (53). There may also
be a mitochondrial isoform (54 –56). To study NO synthase
activity, we used L-NAME, an unselective NO synthase inhibitor. Interestingly, L-NAME was able to reduce the NO levels of
APPsw cells to the levels of control cells (Fig. 1E), showing an
enhanced activity of NOS in APPsw cells. In agreement with
this finding, a rise in nitrosated tyrosines could be detected
(Fig. 1D), which paralleled elevated NO levels (Fig. 1E). To
elucidate which isoform was responsible for the enhanced NO
levels in APPsw cells, we used selective NO synthase inhibitors
for the neuronal NOS and inducible NOS isoforms (57, 58). The
decrease of NO levels in APPsw PC12 cells after exposure to
the neuronal NOS inhibitor N␻-propyl-L-arginine was ⬃9.4%
compared with 4.2% in control cells and 3.7% in APPwt cells.
After exposure to the inducible NOS inhibitor 1400W, the reduction of NO levels was ⬃13.7% in APPsw PC12 cells compared with 3.3% in control cells and 4.6% in APPwt cells (data
not shown). Thus, both isoforms seemed to be involved in the
A␤-induced NO increase in APPsw PC12 cells. We cannot rule
out the involvement of the endothelial NOS isoform in A␤induced NO increase, but this experiment could not be performed due to the lack of commercially available selective
endothelial NOS inhibitors.
Amyloid ␤ Leads to Mitochondrial Damage—Because mitochondria play a key role in the cell death decision, we investigated the effect of the APPsw mutation on mitochondrial function in PC12 cells and HEK cells. Additionally, we have also
analyzed the effects of extracellular A␤1– 42 on mitochondrial
function in PC12 cells.
Mitochondrial membrane potential is a very important
marker for the function of mitochondria (15). A decrease in
mitochondrial membrane potential has been related to cell
death in different cell types. Interestingly, APPsw-bearing
PC12 cells showed a hyperpolarized mitochondrial membrane
potential compared with control cells and APPwt PC12 cells
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FIG. 1. Effects of short and long term exposure to A␤ on apoptotic cell death and intracellular NO levels. Apoptotic cell death of PC12
(A) and HEK cells (B) stably transfected with human APP and extracellular A␤-treated PC12 cells (C). A, unchanged basal apoptosis in APPsw
PC12 cells. B, increase in apoptotic cells in APPwt and APPsw HEK cells compared with control (co) cells (ANOVA: ***, p ⬍ 0.001; *, p ⬍ 0.05 versus
control cells). C, increase in apoptotic cells after treatment with extracellular A␤ for 24 h (ANOVA: **, p ⬍ 0.01 versus untreated PC12 cells). Values
are means ⫾ S.E. from 6 – 8 experiments. D, elevation of nitrotyrosine protein expression in APPsw PC12 cells. Intracellular NO levels of PC12 (E)
and HEK cells (F) stably transfected with human APP and extracellularly A␤-treated PC12 cells (G). E, APPsw PC12 cells exhibited significantly
increased basal NO levels. NOS activity is enhanced in APPsw PC12 cells. NO levels of APP-transfected PC12 cells were determined after a 48-h
incubation in the absence or in the presence of the NO synthase inhibitor L-NAME (20 mM) (ANOVA: ⫹⫹⫹, p ⬍ 0.001; ⫹, p ⬍ 0.05 versus control
PC12 cells; ***, p ⬍ 0.001; **, p ⬍ 0.01 versus untreated APPwt and APPsw PC12 cells). F, basal NO levels are increased in APPsw HEK cells
compared with control HEK cells (ANOVA: ***, p ⬍ 0.001 versus control HEK cells; *, p ⬍ 0.05 versus APPwt HEK cells). G, increase in NO levels
after extracellular A␤ treatment for 24 h (ANOVA: **, p ⬍ 0.01; *, p ⬍ 0.05 versus untreated PC12 cells). Values are means ⫾ S.E. from
6 –14 experiments.
50314
Amyloid ␤, Nitric Oxide, and Mitochondria
(Fig. 2A). However, APPsw HEK cells, which have 20-fold
increased A␤ levels, showed a significantly decreased mitochondrial membrane potential in comparison with control HEK
cells and APPwt HEK cells (Fig. 2B). These findings are in
accordance with the levels of apoptotic cells, which were unchanged in APPsw PC12 cells but significantly increased in
APPsw HEK cells. Treating PC12 cells with extracellular
A␤1– 42 resulted also in a significant depolarization of mitochondrial membrane potential (Fig. 2C) but to a lesser degree than
in APPsw HEK cells. Thus, it appears to be important to
distinguish between acute and chronic A␤ effects on the one
hand and between low and high dose-dependent effects of A␤
on mitochondrial function on the other hand.
Mitochondria are the major source of ATP. Interestingly, not
only APPsw PC12 cells but also APPwt PC12 cells show reduced ATP levels in comparison with control PC12 cells (Fig.
2D). HEK cells bearing the APPsw mutation also have significantly decreased ATP levels in the following order: APPsw ⬍
APPwt ⬍ control cells (Fig. 2E). The ATP reduction is stronger
in APPsw HEK cells than in APPsw PC12 cells, probably indicating a dose-dependent effect of A␤ on ATP levels.
In addition, the extracellular A␤-treated PC12 cells showed
reduced ATP levels (Fig. 2F). As already observed with NO
measurement, the maximal effect was already seen after
exposure to 1 nM A␤1– 42. Again, the ATP reduction was stronger under physiological conditions using APPsw PC12 cells
compared with acute treatment with extracellular A␤ (37
versus 13%).
The Inhibition of ␥-Secretase Leads to a Reduction of NO
Levels and Stabilization of Mitochondrial Function in APPtransfected Cells—To further demonstrate that mitochondrial
damage is due to intracellular A␤ and not due to overexpression of APP, APP-transfected PC12 and HEK cells were treated
with DAPT, a functional ␥-secretase inhibitor that reduces
intracellular A␤ levels without affecting APP expression (59 –
61). In PC12 and HEK cells, DAPT does not show neurotoxicity
by itself. We observed no decrease in MTT reduction after 24,
48, and 72 h of DAPT incubation (data not shown). However,
we could show a strong reduction of secreted A␤1– 40 levels
already after 24 h of DAPT incubation in APPsw PC12 cells and
after 48 h in APPsw HEK cells (Table I). Thus, the reduction of
secreted A␤1– 40 below the level of control cells was very drastic
in APPsw HEK cells, which secreted 20-fold increased A␤ levels compared with APPsw PC12 cells. A similar decrease in
secreted A␤ was also seen in APPwt cells (data not shown). As
already shown by others (62), DAPT also reduces significantly
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FIG. 2. Effects of short and long term exposure to A␤ on mitochondrial membrane potential and ATP levels. Measurement of ␺m in
PC12 (A) and HEK cells (B) stably transfected with human APP and extracellular A␤-treated cells (C). A, hyperpolarization of mitochondrial
membrane potential in APPsw PC12 cells (ANOVA: **, p ⬍ 0.01 versus control PC12 cells; *, p ⬍ 0.05 versus APPwt PC12 cells). B, mitochondrial
membrane potential is significantly decreased in APPwt HEK cells and APPsw HEK cells compared with control HEK cells (ANOVA: ***, p ⬍ 0.001
versus control HEK cells). C, extracellular A␤ treatment for 24 h leads to a decrease of mitochondrial membrane potential in PC12 cells (ANOVA,
***, p ⬍ 0.001; **, p ⬍ 0.01 versus untreated PC12 cells). Values are means ⫾ S.E. from 6 –15 experiments. Determination of ATP levels in PC12
(D) and HEK cells (E) stably transfected with human APP and extracellular A␤-treated PC12 cells (F). D, reduction of ATP levels in APPwt and
APPsw PC12 cells (ANOVA: ***, p ⬍ 0.001 versus control PC12 cells; *, p ⬍ 0.05 versus APPwt PC12 cells). E, ATP levels of APPwt and APPsw
HEK cells are significantly decreased (ANOVA: ***, p ⬍ 0.001; *, p ⬍ 0.05 versus control HEK cells; **, p ⬍ 0.01 versus APPwt HEK cells). F,
extracellular A␤ treatment for 24 h leads to a reduction of ATP levels (ANOVA: ***, p ⬍ 0.001 versus untreated PC12 cells). Values are means ⫾
S.E. from 9 –18 experiments. co, control.
Amyloid ␤, Nitric Oxide, and Mitochondria
50315
FIG. 3. The ␥-secretase inhibitor DAPT reduces NO levels and restores mitochondrial activity. APPsw PC12 cells were incubated for
48 h with DAPT. Afterward, NO levels (A), ATP levels (B), and mitochondrial membrane potential (C) were determined (ANOVA: ***, p ⬍ 0.001;
**, p ⬍ 0.01; *, p ⬍ 0.05 versus untreated APPsw PC12 or APPsw HEK cells). Values are means ⫾ S.E. from six experiments.
known to inhibit the mitochondrial respiratory chain (30).
Therefore, we analyzed the COX activity. Interestingly, APPsw
PC12 cells showed a significantly reduced cytochrome c oxidase activity compared with APPwt and control PC12 cells
(Fig. 6A). Moreover, we measured the mitochondrial membrane potential with the fluorescence dye TMRE after stimulation with different complex inhibitors. Here, we showed
that respiratory chain complexes II, III, IV, and F0F1-ATPase
in APPsw PC12 cells are more vulnerable against mitochondrial membrane potential changes than control PC12 cells,
indicating an impaired mitochondrial respiratory chain (Table II). Additionally, the complexes IV and F0F1-ATPase of
APPwt PC12 cells are more sensitive than control PC12 cells
but are as sensitive as APPsw PC12 cells. Thus, these two
enzyme complexes are especially vulnerable to already very
low A␤ concentrations.
Oxidative Stress Induces Mitochondrial Damage in PC12
Cells Bearing the Swedish APP Mutation—Oxidative stress
might be involved in the pathogenesis of Alzheimer’s disease
(64). Thus, we investigated the effect of a secondary insult,
hydrogen peroxide, which increases cell death in APPsw PC12
cells (9), on mitochondrial membrane potential and ATP levels.
Interestingly, APPsw-bearing PC12 cells showed a significantly decreased mitochondrial membrane potential after exposure to hydrogen peroxide in comparison with APPwt and
control PC12 cells (Fig. 6B). Moreover, the ATP reduction after
hydrogen peroxide exposure was more pronounced in APPsw
and APPwt PC12 cells compared with control PC12 cells, even
in the recovery phase during 2, 4, and 6 h of H2O2 exposure in
a time-dependent manner (Fig. 6C). Thus, mutant cells have
reduced capability to maintain mitochondrial membrane potential and ATP levels after oxidative stress and constantly
showed an ATP deficiency.
The Impact of Mutant APP on Members of the Bcl-2 Family
and on the Release of Mitochondrial Factors—The Bcl-2 family
proteins are important regulators of apoptosis (10). They are
proapoptotic or antiapoptotic in nature. That means that they
induce or inhibit the release of mitochondrial proteins in the
cytosol including cytochrome c, Smac, and AIF. By Western
blot analysis, we investigated the proteins of the Bcl-2 family
as well as Smac and AIF. We have previously shown that a
time-dependent release of cytochrome c was observed after
treatment with hydrogen peroxide, reaching a maximum after
6 h (9). After 4 h of treatment with hydrogen peroxide, there
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intracellular A␤ production in both cell lines. Of note, the
reduction of A␤ levels by the ␥-secretase inhibitor DAPT lead to
a normalization of NO (Fig. 3A) and ATP levels (Fig. 3B) as well
as mitochondrial membrane potential (Fig. 3C) in APPwt and
APPsw PC12 and HEK cells, respectively. Thus, the increased
NO production and mitochondrial damage in APP-transfected
cells were triggered by the production and accumulation of A␤.
Our data suggested that mainly the PC12 cell model represents a very suitable approach to elucidate AD-specific cell
death pathways under physiological conditions that are the
most relevant for the in vivo situation in man because PC12
cells bearing the APPsw mutation secrete A␤ levels within the
picomolar range. For this reason, we made all of the following
experiments with this cell model.
APPsw PC12 Cells Have an Increased Amount of Mitochondria and Show Presence of APP in Mitochondria—To exclude
the possibility that ATP levels are reduced in APP-transfected
PC12 cells due to a loss of mitochondria, we determined the
amount of mitochondria in APP-transfected cells using the
mitochondrial dye Mitotracker Red and confirmed co-localization with the mitochondrial membrane potential dye Rhodamine 123 (Fig. 4, A–C). Mitotracker Red selectively stains mitochondria independently from the mitochondrial membrane
potential. Interestingly, APPsw PC12 cells contained more mitochondria than APPwt and control PC12 cells (Fig. 4, D–G).
Thus, the drop of ATP levels in APPsw PC12 cells was due to
mitochondrial dysfunction and not due to the loss of mitochondria. Moreover, the up-regulation of mitochondria seems not to
be able to compensate for mitochondrial dysfunction with regard to ATP levels.
Recently, it has been shown that APP is also targeted to
mitochondria (63) in addition to its localization in the endoplasmic reticulum and the plasma membrane. In this study, transiently transfected cells were used (63). Interestingly, we also
observed the presence of APP in mitochondria of stably transfected APPwt and APPsw PC12 cells (Fig. 5). The purity of
subcellular fractions was established by Western blot analysis
of various fractions using antibodies to Na⫹/K⫹-ATPase
(plasma membrane-specific), cytochrome c oxidase subunit 4
(mitochondria-specific), and calreticulin (endoplasmic reticulum-specific) as markers. Further investigations have to be
done to further elucidate the role of APP in the mitochondrion.
PC12 Cells Bearing the Swedish APP Mutation Showed an
Impairment of the Mitochondrial Respiratory Chain—NO is
50316
Amyloid ␤, Nitric Oxide, and Mitochondria
FIG. 5. Western blot analysis of human APP expression in mitochondria
of transfected PC12 cells. A, Western
blot analysis of APP (arrows indicate mature and immature human APP695), actin, and COX4 in mitochondrial and cytosolic fraction. B, Western blot analysis of
Na⫹/K⫹-ATPase and calreticulin in mitochondrial fraction and pellet. The pellet
contains all of the cellular compartments
with the exception of mitochondrial and
cytosolic fraction. APP is present in mitochondria of APPwt and APPsw PC12
cells. The purity of subcellular fractions
was evaluated by Western blot analysis of
various subcellular fractions using antibodies to Na⫹/K⫹-ATPase (plasma membrane-specific), COX4 (mitochondria-specific), and calreticulin (endoplasmic
reticulum-specific) as markers. Actin
serves as a marker for equal loading.
was an enhanced release of Smac observed in APPsw PC12
cells (Fig. 7). AIF, a protein that causes nuclear condensation
and DNA fragmentation, was not released by the mitochondria
after 6 h of hydrogen peroxide incubation (Fig. 7); however, we
observed a higher AIF expression in the mitochondria of
APPsw PC12 cells at this time point. Interestingly, we found
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FIG. 4. Staining of mitochondria in APP-transfected PC12 cells. A, APPsw PC12 cells were stained with Mitotracker Red. B, the same cell
was co-stained with Rhodamine 123. C, co-localization of Mitotracker Red and Rhodamine 123. D, control PC12 cells. E, APPwt PC12 cells. F,
APPsw PC12 cells were stained with Mitotracker Red. G, Mitotracker Red fluorescence of APPsw PC12 cells is enhanced. APPsw PC12 cells tend
to have more mitochondria than APPwt and control PC12 cells (unpaired Student’s t test: *, p ⬍ 0.05 versus control PC12 cells). Values are means ⫾
S.E. from six experiments.
Amyloid ␤, Nitric Oxide, and Mitochondria
50317
TABLE II
Reduction of mitochondrial membrane potential after stimulation
with respiratory chain complex inhibitors compared to
control PC12 cells (control ⫽ 100%)
One way ANOVA posthoc Tukeys Multiple Comparison Test. Values
are means ⫾ S.E. from 4 – 8 experiments.
Inhibition of respiratory chain
Complex I by rotenone (2 ␮M)
Complex II by thenoyl-trifluoroacetone
(10 ␮M)
Complex III by antimycin (2 ␮M)
Complex IV by sodium azide (10 mM)
Complex V by oligomycin (10 ␮M)
APPwt
APPsw
n.s.
n.s.
n.s.
⫹36.31%b
n.s.
⫹119.8%a
⫹27.0%b
⫹128.61%a
⫹131.0%a
⫹23.5%c
p ⬍ 0.001 versus control cells (n.s., not significant).
p ⬍ 0.05.
c
p ⬍ 0.01.
a
b
AIF release after 24 h of hydrogen peroxide incubation (data
not shown).
Concerning the Bcl-2 family, we found a reduced amount of
the cytosolic anti-apoptotic protein Bcl-xL in APPsw cells under
base-line conditions and after exposure to hydrogen peroxide
but only minor changes in Bax protein content (Fig. 7). Thus,
the Bcl-xL/Bax ratio was decreased in APPsw PC12 cells under
basal conditions (control cells, 1.0; APPwt cells, 0.86; APPsw
cells, 0.49), which might possibly explain the enhanced vulnerability to cell death after oxidative stress.
APP tg Mice Showed a Decreased Mitochondrial Membrane
Potential and Reduced ATP Levels—To understand the in vivo
relevance of our in vitro findings, we analyzed mitochondrial
membrane potential and ATP levels in dissociated neurons of
3-month-old APP tg mice. Interestingly, the mitochondrial
membrane potential in APP tg mice was significantly decreased under basal conditions compared with littermate
non-tg control mice (Fig. 8). Additionally, dissociated neurons
of tg APP mice showed reduced basal ATP levels (Fig. 8).
DISCUSSION
AD is associated with multiple lesions including changes in
energy metabolism and altered mitochondrial structure and/or
function in the brain. However, the precise mechanisms of
mitochondrial pathology in AD are not clear.
Our study clearly demonstrates that A␤ induces mitochondrial adaptation and failure in a very vulnerable and dose-dependent pattern, which additionally overreacts to secondary
insult, and that NO plays an important role in these processes.
Several other studies already indicate that A␤ impairs mitochondrial function (18, 20, 65). However, nearly all of the
studies used synthetic A␤ peptides in the micromolar range,
many orders of magnitude over physiological levels, and cells or
even isolated mitochondria were exposed only to extracellular
A␤. By contrast, our PC12 cell model represents a very valuable approach to investigate AD-specific cell death pathways by
studying A␤ levels within the high picomolar range (7). This
cell model attempts to mimic physiological conditions in man
that are relevant for sporadic AD patients (66, 67). The comparison between transfected PC12 cells and HEK cells that
exhibit A␤ levels within the low nanomolar range further offered the possibility to compare effects of A␤ on mitochondrial
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FIG. 6. Reduced cytochrome c oxidase activity and increased vulnerability to secondary insult in APP-transfected PC12 cells. A,
cytochrome c oxidase activity is significantly reduced in APPsw PC12 cells (ANOVA: *, p ⬍ 0.05 versus control PC12 cells). B, APPsw PC12 cells
show a significantly decreased mitochondrial membrane potential after exposure to hydrogen peroxide for 2, 4, and 6 h compared with APPwt and
control PC12 cells (ANOVA: **, p ⬍ 0.01; *, p ⬍ 0.05 versus control PC12 cells). C, the ATP reduction after hydrogen peroxide incubation is more
pronounced in APPsw PC12 cells in comparison with APPwt and control PC12 cells (ANOVA: ***, p ⬍ 0.001; **, p ⬍ 0.01; *, p ⬍ 0.05 versus control
PC12 cells or APPwt PC12 cells). Values are means ⫾ S.E. from 5–12 experiments. co, control.
50318
Amyloid ␤, Nitric Oxide, and Mitochondria
FIG. 8. Mitochondrial membrane potential and ATP levels of
APP tg mice. Brain cells from APP tg mice exhibit a significantly
decreased mitochondrial membrane potential and reduced ATP levels.
Two-way ANOVA revealed a significant effect of transgene (*, p ⬍ 0.05)
and between mitochondrial membrane potential and ATP levels (***,
p ⬍ 0.001). Values are means ⫾ S.E. from 6 –12 experiments.
function in a dose-dependent way. As a result, the latter cell
model may reflect increased cellular stress evoked by high
pathological levels of A␤, a situation that might be present in
familial AD patients (66). Moreover, these two cell models
allowed us to study not only effects induced by extracellular A␤
but also the effects of intracellularly generated A␤ and/or other
products of the APP-processing pathway, e.g. APP intracellular
domain, on mitochondrial function. This is an important advantage over extracellular A␤ treatment, because strong evidence indicates the important role of the intracellular biology of
A␤ in the pathogenesis of AD.
Our data suggested that NO plays an important role in
A␤-induced mitochondrial dysfunction and cell death and that
intracellularly produced A␤ is specifically relevant. The mitochondrial respiratory chain is sensitive to both NO- and peroxynitrite-mediated damage (30 –32). In accordance with these
findings, APPsw PC12 cells showed reduced cytochrome c oxidase activity that is probably due to a direct inhibitory effect of
NO, whereas the complexes II, III, and V in APPsw cells
seemed to be impaired by peroxynitrite. Importantly, APPwt
PC12 cells also showed an impairment of cytochrome c oxidase
and complex V. Thus, these two complexes are especially susceptible to already low A␤ levels. Recently, it has been shown
that APP and A␤ are targeted to mitochondria (63, 68). In
accordance, we observed the presence of APP in the mitochondria of APPwt and APPsw PC12 cells. This presence of APP in
mitochondria might be involved in the impairment of the mi-
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FIG. 7. Changes in mitochondrial factors regulating apoptosis after secondary insult. A, Western blot analysis of Bcl-xL, Bax, Smac, and
AIF in mitochondrial and cytosolic fractions of H2O2-treated PC12 cells. After 4 h, we found an enhanced Smac release in APPsw PC12 cells.
Moreover, we observed a reduced amount of the cytosolic anti-apoptotic protein Bcl-xL in APPsw PC12 cells under base-line conditions and after
exposure to hydrogen peroxide. B, densitometric analysis of cytosolic fractions (two-way ANOVA Bcl-xL: ***, p ⬍ 0.001 between cell lines; Smac:
***, p ⬍ 0.001 between time course; ***p ⬍ 0.001 between cell line). Values are means ⫾ S.E. from 3–9 experiments. co, control.
Amyloid ␤, Nitric Oxide, and Mitochondria
sequence of events linking A␤, NO production, ATP depletion,
and mitochondrial membrane potential with caspase pathway
and neuronal cell loss.
Under physiological conditions, the mitochondrial membrane potential was estimated at ⫺150 to ⫺180 mV with respect to the cytosol. Following acute stimulation with extracellular A␤, we and other groups (72) found a slight decrease in
mitochondrial membrane potential at rather high non-physiological A␤ concentrations. This might have resulted from an
inhibition of respiratory chain complexes by increased NO levels leading to a drop in the electron transport and consequently
in proton extrusion.
In contrast, APPsw-transfected PC12 cells, which chronically secrete A␤ levels in the picomolar range, showed activation of a defense response in an attempt to maintain cellular
function and viability. After an initial decrease of mitochondrial membrane potential, chronic inhibition of the respiratory
chain by increased NO levels resulted in hyperpolarization of
mitochondrial membrane potential, probably due to an increase in the rate of glycolysis followed by the entering of
glycolytic ATP into the mitochondria (73–75). Consistent with
this finding, we observed no increased basal apoptosis in
APPsw PC12 cells. Interestingly, APPsw HEK cells, which
express chronically high A␤ levels, have no possibility to adapt.
This is probably due to the prolonged increase in the generation
of superoxide anions in the presence of high A␤ levels resulting
in the formation of peroxynitrite and thereby facilitating the
opening of the permeability transition pore (76, 77). This finally
leads to the collapse of mitochondrial membrane potential and
the release of mitochondrial proteins including cytochrome c
and Smac that in turn activates caspase cascade and cell death.
Consistently, we found increased basal apoptotic levels in
APPsw HEK cells. A similar situation exists in the brains of
APP tg mice at an age of 3 months, which have detectable high
A␤ levels in the brain but before the onset of plaque formation.
The brain cells of these mice showed a significantly decreased
mitochondrial membrane potential. Thus, we have distinct effects of A␤ on mitochondrial function dependent on acute or
chronic exposure and on low and high concentration levels of
the peptide. These different effects might play a role in sporadic
and familial forms of the disease.
However, when APPsw PC12 cells were stressed with an
AD-relevant secondary insult, e.g. oxidative stress, the cells
were no longer able to compensate for mitochondrial dyshomeostasis and the mitochondrial membrane potential significantly decreased. In addition, the ATP reduction after oxidative stress was stronger in APPsw PC12 cells than in control
cells. Thus, the increased A␤ production at physiological levels
enhanced the vulnerability against mitochondrial dysfunction
after oxidative stress in APPsw PC12 cells, a process that
might be also relevant in sporadic AD. As we could show, a shift
in the Bcl-xL/Bax ratio toward Bax might be involved in the
higher sensitivity of APPsw cells to mitochondrial failure after
oxidative stress. Secondary stress insult finally lead to the
release of mitochondrial proteins, e.g. Smac and AIF, which
play a role within the cell death cascade (78, 79). The increased
Smac release in APPsw cells might explain the higher caspase
9 and caspase 3 activity of APPsw PC12 cells as previously
demonstrated (9). In contrast, AIF seems to play only an important role in the late phase of apoptosis in APPsw PC12 cells.
Thus, one can speculate that, also in humans, increased A␤
accumulation and associated mitochondrial toxicity are contributing factors in the pathogenesis of AD syndromes. At the
beginning, the damaging effects of low physiological concentrations of A␤ could be overcome by an adaptive response. However, when age-related secondary stress occurs, pronounced
Downloaded from www.jbc.org by on January 29, 2009
tochondrial respiratory chain. Thus, mitochondrial dysfunction
can be induced on the one hand via enhanced NO levels and on
the other hand because of a direct effect of A␤ on mitochondria
(69), although we can not exclude additional effects of APP
itself or other intracellular APP-processing products such as
APP intracellular domain on mitochondria.
The reduced cytochrome c oxidase activity in APPsw PC12
cells is consistent with the observation that basal ATP levels of
APPsw PC12 cells were significantly reduced compared with
control cells. Interestingly, APPwt PC12 cells also show reduced ATP levels but to a lesser extent than APPsw PC12 cells.
We suggest that the very low A␤ levels in APPwt PC12 cells are
sufficient to reduce ATP levels but that this depletion of ATP
does not induce cell death per se, indicating that APPsw PC12
cells are able to compensate for this deficit under normal conditions. Only after treatment with a secondary insult (in this
case oxidative stress), APPsw PC12 cells were no longer able to
maintain cellular function and cell death was increased. Afterward, extracellular A␤ treatment showed only weak effects on
ATP reduction, suggesting again that A␤ exhibits its effects on
mitochondrial function mostly by intracellular effects. In addition, the ATP reduction in APPsw HEK cells was stronger than
in APPsw PC12 cells, showing a dose-dependent effect of A␤ on
ATP levels. To investigate whether APP or A␤ is mainly responsible for the mitochondrial damage, the intracellular A␤
production was inhibited by the ␥-secretase inhibitor DAPT,
which is known to reduce intracellular A␤ levels without affecting APP expression (59 – 61). Our results showed that the
DAPT-mediated inhibition of A␤ production lead to a stabilization of mitochondrial function with regard to both a pathogenic
hyperpolarization and depolarization of mitochondrial membrane potential in APPsw PC12 cells and APPsw HEK cells,
respectively, and to a decrease in NO levels in APPsw PC12 as
well as in HEK cells. This finding clearly indicated that increased NO production and mitochondrial dysfunction resulted
from increased A␤ levels in APP-transfected PC12 and HEK
cells. Our model further strengthened the thesis that A␤
mainly mediates the toxic intracellular effects. However, mitochondrial function did not recover completely to normal levels.
This might be due to irreparable damage of the mitochondria
by long term expression of A␤ or to APP still persisting in the
mitochondrion that may directly interfere with mitochondrial
respiratory chain. Very importantly, in 3-month-old APP tg
mice, reduced ATP levels can also be observed under basal
conditions showing the in vivo relevance of our findings. Of
note, the 3 month-old mice exhibited no plaques but already
detectable A␤ levels in the brain (39), probably emphasizing
again the very important role of intracellular A␤ in ATP reduction. In 12-month-old amyloid plaque-bearing transgenic mice
bearing the Swedish APP mutation, reduced cytochrome c oxidase activity and reduced ATP levels were also found (63). Thus,
A␤ impairs the energy metabolism of mitochondria in different
AD models, possibly as a very early event in the pathogenesis of
the disease, as has been shown for AD patients (70).
We could further exclude the possibility that the reduction of
ATP levels is due to a reduced number of mitochondria in
APPsw PC12 cells, because APPsw transfection rather increased the amount of mitochondria, possibly due to an adaptation process, in response to the increase in NO and reduction
of ATP, respectively. This is in accordance with other findings
demonstrating a stimulation of the mitochondrial biogenesis by
NO (71). Nevertheless, the increased number of mitochondria
was not sufficient to compensate the reduced ATP levels in
APPsw PC12 cells.
Based on our data and the knowledge regarding the effects of
NO on mitochondrial function (30), we proposed a hypothetical
50319
Amyloid ␤, Nitric Oxide, and Mitochondria
50320
mitochondrial impairment might lead to the induction of cell
death processes, whereas in familial AD, high A␤ load might be
directly responsible for mitochondrial and cellular dysfunction.
In summary, we showed novel and distinct actions of A␤ on
mitochondria in vitro and in vivo that may contribute to the
pathogenic outcome.
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