 |
 Previous Article | Next Article 
The Journal of Neuroscience, September 1, 1999, 19(17):7394-7404
Mitochondrial Depolarization Is Not Required for Neuronal
Apoptosis
Aaron J.
Krohn1,
Tanja
Wahlbrink1, and
Jochen H. M.
Prehn1, 2
1 Interdisciplinary Center for Clinical Research (IZKF),
Junior Research Group "Apoptosis and Cell Death," Westphalian
Wilhelms-University, D-48149 Münster, Germany, and
2 Department of Pharmacology and Toxicology,
Philipps-University, D-35032 Marburg, Germany
 |
ABSTRACT |
Mitochondria are sites of cellular energy production but may also
influence life and death decisions by initiating or inhibiting cell
death. Mitochondrial depolarization and the subsequent release of
pro-apoptotic factors have been suggested to be required for the
activation of a cell death program in some forms of neuronal apoptosis.
We induced apoptosis in cultured rat hippocampal neurons by exposure to
the protein kinase inhibitor staurosporine (STS) (300 nM).
The time course of mitochondrial membrane potential (  m) during apoptosis was examined using the
probe tetramethylrhodamine ethyl ester (TMRE). Cells exhibited no
decrease in TMRE fluorescence, indicative of mitochondrial
depolarization, up to 8 hr after STS exposure. Rather, baseline TMRE
fluorescence remained unchanged up to 2 hr and thereafter actually
increased significantly. Throughout this time period, the mitochondria
could also be depolarized with the protonophore carbonyl cyanide
p-trifluoromethoxy-phenylhydrazone (FCCP, 0.1 µM), exhibiting the same relative magnitude of
fluorescence release (unquenching) as controls. Even after 16 hr of
staurosporine treatment, neurons that showed signs of nuclear apoptosis
maintained m and could be depolarized with FCCP. In
contrast, caspase-3-like activity had increased roughly sevenfold by 2 hr and >20-fold by 8 hr. Double-labeling of hippocampal neurons with
the potential-sensitive probe Mitotracker Red Chloromethyl X-Rosamine
and an antibody to cytochrome c demonstrated at the subcellular level
that mitochondrial cytochrome c release also occurred in the absence of
mitochondrial depolarization. These data suggest that mitochondrial
depolarization is not a decisive event in neuronal apoptosis.
Key words:
programmed cell death; mitochondrial membrane potential
(m); cytochrome c; permeability
transition pore (PTP); caspases; staurosporine (STS); tetramethylrhodamine ethyl ester (TMRE); carbonyl cyanide
p-trifluoromethoxy-phenylhydrazone (FCCP)
| |
INTRODUCTION |
It has become increasingly clear
that the activation of a cell death program may be responsible for
pathophysiological cell death in neurodegenerative disorders, including
Alzheimer's disease (Thompson, 1995 ). Known elements of this program
include members of the bcl-2 gene family, Apaf-1, and proteases of
the caspase family (Cohen, 1997; Reed, 1997a; Salvesen and Dixit, 1997;
Zou et al., 1997).
Once thought to be merely the center of cellular ATP production,
mitochondria are increasingly implicated as sensors and executioners in
the cell's decision to live or die (Kroemer et al., 1997; Reed, 1997b;
Murphy et al., 1999) and may even influence the mode of cell death
chosen, necrosis or apoptosis, depending on their functional state
(Nicotera and Leist, 1997; Tsujimoto, 1997). It has been suggested that
mitochondria are capable of releasing pro-apoptotic factors into the
cytosol, including apoptosis-inducing factor (AIF), which has been
shown to induce chromatin condensation and oligonucleosomal DNA
fragmentation in isolated nuclei (Susin et al., 1996, 1999; Zamzami et
al., 1996a), and cytochrome c, which can activate, with the help of
Apaf-1 and (d)ATP, procaspase-9 and the caspase cascade (Li et al.,
1997; Zou et al., 1997).
What remains as yet unresolved is the mechanism by which pro-apoptotic
factors are released into the cytosol. One candidate is the so-called
mitochondrial permeability transition pore (PTP) (Kroemer et al., 1997;
Marzo et al., 1998). The PTP is triggered by an increase in matrix
Ca2+, pro-oxidants, and mitochondrial
depolarization and appears to be nonspecific and permeable to solutes
under 1.5 kDa (Zoratti and Szabo, 1995). Because it results in inner
membrane permeability to protons, PTP is associated with a decrease in
mitochondrial transmembrane potential (m)
(Zoratti and Szabo, 1994; Zamzami et al., 1996a).
Because cytochrome c has a molecular weight of 15 kDa, the opening of a
PTP is probably not sufficient to release cytochrome c directly but
could trigger cytochrome c release by causing mitochondrial swelling
and/or rupture of the outer mitochondrial membrane (Vander Heiden et
al., 1997).
A reduction in m has been observed in a
number of models of apoptosis (Ankarcrona et al., 1995; Zamzami et al.,
1996b; Boise and Thompson, 1997; Wadia et al., 1998; Heiskanen et al.,
1999). On the other hand, however, studies in non-neuronal cells have suggested that this is not a primary event in apoptosis (Kluck et al.,
1997; Vander Heiden et al., 1997; Yang et al., 1997; Bossy-Wetzel et
al., 1998; Yoshida et al., 1998). Thus, although the importance of
mitochondria in programmed cell death (PCD) seems fairly clear with respect to the release of pro-apoptotic factors, the events preceding this remain unresolved. Staurosporine (STS), a potent inhibitor of survival protein kinases (Bertrand et al., 1994), is
widely used to activate PCD in neuronal and non-neuronal cells (Falcieri et al., 1993; Koh et al., 1995; Prehn et al., 1997). We
examined the importance of mitochondrial depolarization in STS-induced
apoptosis of cultured rat hippocampal neurons using the
potential-dependent probes tetramethylrhodamine ethyl ester (TMRE) and
Mitotracker Red Chloromethyl X-Rosamine (CMXRos) in conjunction with
digital video and immunofluorescence microscopy.
| |
MATERIALS AND METHODS |
Cell culture. Primary cultures of hippocampal neurons
were prepared from neonatal (P1) Fischer 344 rats according to Prehn (1998). Dissected hippocampi were incubated for 20 min at 37°C in
Leibovitz L-15 medium (Life Technologies, Eggenstein, Germany) containing 0.1% papain. Afterward, the medium was removed, and the
cells were suspended by gentle trituration in MEM supplemented with
10% NU-serum, 2% B-27 supplement (50× concentrate), 2 mM L-glutamine, 20 mM D-glucose, 26.2 mM sodium bicarbonate, 100 U/ml penicillin, and 100 µg/ml
streptomycin (Life Technologies). The suspension was layered over
medium containing 10 mg/ml trypsin inhibitor and centrifuged for 10 min
at 600 rpm. The cells were then resuspended, plated, and incubated in
MEM at 37°C in an atmosphere of 95% air and 5% carbon dioxide. For
imaging studies, cells were grown on poly-L-lysine-coated
glass coverslips that had been placed into 35 mm Petri dishes (Falcon,
Heidelberg, Germany). For immunofluorescence microscopy experiments,
cells were plated onto eight-well tissue culture slides
(Becton-Dickinson, Heidelberg, Germany). In all other cases, neurons
were plated onto poly-L-lysine-coated 24-well plates (Nunc,
Hamburg, Germany). After 24 hr in vitro, cultures were
treated with the anti-proliferation agent cytosine
 -arabinofuranoside (CAF) (1 µM; Sigma,
Deisenhofen, Germany). After an additional 24 hr, the cells were given
fresh CAF-containing medium. Experiments were performed on 8- to
10-d-old cultures. Animal care followed official government guidelines.
Induction of apoptosis. We induced apoptosis in rat
hippocampal neurons by exposure to the protein kinase inhibitor STS.
STS-induced cell death is a commonly used model in the study of
apoptosis in both neuronal and non-neuronal cell types (Falcieri et
al., 1993; Bertrand et al., 1994; Koh et al., 1995; Prehn et al.,
1997); is characterized by shrinkage of the cell body, membrane
blebbing, chromatin condensation, nuclear pyknosis, and positive
labeling of 3'-OH-DNA ends using the terminal deoxynucleotidyl
transferase-based dUTP-digoxigenin nick-end labeling (TUNEL) technique;
and can be reduced by 24 hr pretreatment with the protein synthesis
inhibitor cycloheximide or the G1/S cell cycle
inhibitor mimosine, as well as by caspase inhibition (Koh et al., 1995;
Prehn et al., 1997; Krohn et al., 1998). To induce apoptosis,
staurosporine (Sigma; 1 mM stock in DMSO) was added to the
culture medium to a final concentration of 300 nM, a
concentration that has been shown to induce rapid apoptotic cell death
in >75% of cultured rat hippocampal neurons (Prehn et al., 1997).
Controls were exposed to an equal volume of the vehicle.
Estimation of mitochondrial membrane potential
(m). TMRE is a cationic, lipophilic dye
that accumulates in the negatively charged mitochondrial matrix
according to the Nernst equation potential (Ehrenberg et al., 1988). A
TMRE (Molecular Probes, Leiden, The Netherlands) stock was prepared at
a concentration of 10 mg/ml in DMSO and stored at  20°C. Working
stocks of 1 mg/ml were made up fresh in distilled water. For estimation
of m, cells were incubated with 100 nM TMRE for 15 min at room temperature in HEPES-buffered
saline (HBS) consisting of (in mM): 144 NaCl, 10 HEPES, 2 CaCl2, 1 MgCl2, 5 KCl, 10 D-glucose; 320 mOsm; pH 7.4. The dye was present in the
buffer during the entire course of the experiment. TMRE fluorescence
was then measured using a fluorescence microscope (Axiovert 100 inverted-stage microscope; Zeiss, Oberkochen, Germany) with a 40×
fluorescence objective and attenuated UV illumination from a 75 W xenon
arc. Optics were as follows: excitation, 490 nm; dichroic mirror, 505 nm; and emission, >510 nm. Under these conditions, autofluorescence
was negligible. Images were collected every 15 sec using an intensified
CCD camera (C 2400-87, Hamamatsu, Herrsching, Germany) with the camera
controller gain set to 2.5. Sixteen frames were averaged for each
image, which was digitized as 256 × 256 eight-bit pixels. A
background image was taken before each experiment and was later
subtracted from the images. Data were analyzed using Argus-50 software
(Hamamatsu). Neurons were recognized by morphology as well as by their
position in a higher plane of focus than astrocytes; they generally
showed considerably lower basal TMRE fluorescence. Fluorescence data, which reflect the average pixel intensity obtained from the neuronal soma excluding the nucleus, are expressed in arbitrary fluorescence units (FlU).
Baseline TMRE fluorescence was measured for the first 5 min of each
experiment. At that point, the cells were exposed to the protonophore
carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP)
(0.1 µM in TMRE-containing HBS; Sigma). FCCP
depolarizes mitochondria by abolishing the proton gradient across the
inner mitochondrial membrane (Gunter and Pfeiffer, 1990; Prehn et al., 1994). After 5 min of exposure, the FCCP-containing buffer was replaced
with fresh TMRE-containing HBS, allowing the cells to recover
m.
For some experiments, the nuclei of cells were imaged after estimation
of m. The culture medium was returned, and
cultures were incubated with the cell-permeant, live-cell nucleic acid stain SYTO 16 (1 mM stock in DMSO; Molecular Probes) at a
concentration of 1 µM for 30 min at 37°C (Frey, 1995).
The medium was then aspirated, the cultures were washed in HBS, and the
nuclear fluorescence was acquired using the above-mentioned imaging
system. Apoptotic nuclei were distinguished by increased SYTO 16 fluorescence, indicative of chromatin condensation (Frey, 1995).
Mitotracker Red CMXRos and cytochrome c double-labeling.
After the indicated length of exposure to 300 nM STS,
hippocampal cultures were incubated with 50 nM Mitotracker
Red CMXRos (Molecular Probes) for 30 min at 37°C in culture medium.
Similarly to TMRE, this dye is taken up by mitochondria as a result of
m (Susin et al., 1999). After uptake,
CMXRos forms thiol conjugates with cell peptides, inhibiting release of
the dye from mitochondria and enabling fixation of cells while
retaining the dye. After incubation with CMXRos, cultures were washed
with PBS, fixed for 20 min at 37°C with freshly made 4% formaldehyde
in PBS, and permeabilized with 0.1% Triton X-100 for 3 min at 4°C.
After permeabilization, the cultures were washed again, then incubated
in blocking buffer (2% low-fat milk powder, 2% bovine serum albumin,
0.1% Tween 20, in PBS, pH 7.4) for 1 hr at room temperature. The
primary antibody (anti-cytochrome c, clone 6H2.B4, PharMingen, San
Diego, CA), which recognizes the native form of cytochrome c, was then
added at a concentration of 10 µg/ml for 2 hr at room temperature.
After washing, biotin-conjugated anti-mouse IgG (1:1000; Vector
Laboratories, Burlingame, CA) was added for 1 hr at room temperature,
followed by a streptavidin-Oregon Green conjugate (1 µg/ml, 20 min,
Molecular Probes). CMXRos and Oregon Green fluorescence were observed
using an Eclipse TE300 inverted microscope and either a 20× dry or
100× oil immersion objective (Nikon, Düsseldorf, Germany) with
the following optics: for CMXRos, excitation, 510-560 nm; dichroic mirror, 575 nm; emission, >590 nm; for Oregon Green, excitation, 465-495 nm; dichroic mirror, 505 nm; emission, 515-555 nm. Digital images of equal exposure for control and STS-treated cultures were
acquired with a SPOT-2 camera (Diagnostic Instruments, Sterling Heights, MI) using Metamorph software (Universal Imaging Corporation, West Chester, PA). The images were deconvoluted using No Neighbor Deblurring software (kindly provided by Dr. B. Lindemann, University of
Saarbrücken, Germany), which applies the algorithm of Monck et
al. (1992) to reduce image background haze attributable to light
originating from unsharp areas of the specimen.
Assessment of mitochondrial mass: quantitation of Mitotracker
Green FM and nonyl acridine orange fluorescence. Mitotracker Green
FM (MTGFM) is a mitochondrion-selective probe that becomes fluorescent
in the lipid environment of mitochondria. MTGFM contains a
thiol-reactive chloromethyl moiety, resulting in stable peptide and
protein conjugates after accumulation in mitochondria. Unlike TMRE and
CMXRos, uptake of this probe is less dependent on
m (see Table 2 and manufacturer's
information), thus allowing estimation of mitochondrial mass in both
live and fixed cells (Metivier et al., 1998). A 1 mM stock
solution of Mitotracker Green FM (Molecular Probes) was made in DMSO.
At the indicated time points after addition of STS, the stock solution
was diluted in the culture medium to a final concentration of 200 nM, and the cultures were incubated for 30 min at 37°C.
The cultures were then rinsed twice with PBS and extracted with 200 µl lysis buffer (10% SDS, 0.1 M Tris, pH 7.5). Extract
fluorescence was then measured using the fluorescent plate reader
described below, with an excitation wavelength of 485 nm and an
emission wavelength of 530 nm. Nonyl acridine orange (NAO) accumulates
in mitochondria by binding to cardiolipin and has been used previously
to measure changes in mitochondrial mass (Cossarizza et al., 1995;
Mancini et al., 1997; Metivier et al., 1998). Experiments with NAO
were performed as with MTGFM, using a dye concentration of 1 µg/ml. Values are expressed as fluorescence units per microgram
protein. Protein content was determined using a Pierce BCA Micro
Protein Assay Kit.
Modulation of plasma membrane potential. The influence of
plasma membrane potential on mitochondrial TMRE uptake was abolished by
incubation in high K+ buffer. Hippocampal
neurons were first treated with 300 nM STS or vehicle for 6 hr. After this, the medium was exchanged for a
Ca2+-free HBS containing either 5 or 50 mM KCl, in which the cells were incubated for 5 min before
the addition of 100 nM TMRE. After a 10 min loading period,
cellular TMRE fluorescence was acquired with the aforementioned SPOT-2
camera system, and fluorescence intensities were analyzed using
Metamorph software.
Measurement of caspase-3-like activity. After the indicated
lengths of exposure to staurosporine, the culture medium was aspirated, the cultures were washed with PBS, and the cells were lysed with 200 µl of lysis buffer [10 mM HEPES, pH 7.4, 42 mM KCl, 5 mM MgCl2, 1 mM PMSF, 0.1 mM EDTA, 0.1 mM EGTA,
1 mM dithiothreitol (DTT), 1 µg/ml pepstatin A, 1 µg/ml
leupeptin, 5 µg/ml aprotinin, 0.5% 3-(3-cholamidopropyldimethylammonio)-1-propane sulfonate (CHAPS)]. Fifty microliters of this extract were added to 150 µl of reaction buffer (25 mM HEPES, 1 mM EDTA, 0.1% CHAPS,
10% sucrose, 3 mM DTT, pH 7.5), which was supplemented
with 10 µM acetyl-DEVD-aminomethylcoumarin (Ac-DEVD-AMC)
(Alexis, Grünstetten, Germany), a fluorogenic substrate for
caspase-3 and related proteases, including caspase-1, -4, -6, -7, and
-8 (Talanian et al., 1997; Garcia-Calvo et al., 1999). Production of
fluorescent AMC was monitored for 120 min at room temperature using a
fluorescent plate reader (FL 500, Biotek, Hamburg, Germany) (excitation
380 nm, emission 460 nm). Fluorescence of blanks containing no cellular
extracts were subtracted from the values. Caspase-3-like activity is
expressed as change in fluorescence units per 120 min per microgram
protein. Total protein content, determined as above, did not change
significantly up to 12 hr into STS exposure.
Nuclear changes. To observe nuclear changes occurring during
apoptosis, the chromatin-specific dye Hoechst 33258 (Sigma) was used.
Cultures were fixed for 10 min with 4% formaldehyde in PBS at 37°C,
then permeabilized by treatment with a 19:1 mixture of ethanol/acetic
acid for 15 min at 20°C. After being washed with PBS, the cells
were stained with 1 µg/ml Hoechst 33258 in PBS for 20 min at room
temperature and then washed again. Hoechst staining was viewed with an
Axiovert 135 fluorescence microscope (Zeiss) using a 40× fluorescence
objective, with UV illumination from a 50 W xenon arc. Optics were as
follows: excitation, 365 nm; dichroic mirror, 395 nm; and emission, 420 nm. Nuclei of control cells appeared oval, with a septate pattern of
blue fluorescence. During STS-induced apoptosis, nuclei displayed
increased brightness, indicative of chromatin condensation, or
fragmented into smaller bodies. Apoptotic and nonapoptotic nuclei were
counted in 10 randomly chosen subfields per culture well.
Inhibitors of mitochondrial permeability transition. The
immune suppressant cyclosporine A inhibits the opening of the
mitochondrial PTP by binding to cyclophilin D and bongkrekic
acid by stabilizing the adenine nucleotide translocator in the
matrix-facing conformation (Zoratti and Szabo, 1995; Murphy et al.,
1999). Hippocampal neurons were pretreated for 1 hr with cyclosporine A
(10 µM) or bongkrekic acid (50 µM), and 300 nM STS was added to the culture medium. After 24 hr
incubation, apoptotic degeneration was assessed by Hoechst staining
(see above).
Statistics. Where not noted otherwise, data are presented as
means ± SEM. For statistical comparison of viability and caspase activity data, one-way ANOVA followed by Tukey's test were used. Imaging data were analyzed using the nonparametric Kruskal-Wallis test, followed by pair-wise comparisons using the Mann Whitney U test. p values <0.05 were considered to be
statistically significant.
| |
RESULTS |
Estimation of mitochondrial membrane potential
Exposure of rat hippocampal neurons to STS in a concentration
range from 10 to 1000 nM induces apoptosis (Prehn et al.,
1997; Krohn et al., 1998). In this study, we used an STS concentration of 300 nM, which results in rapid and extensive neuronal
cell death in the cultures (Fig. 1)
(Prehn et al., 1997). To relate changes in
m to these apoptosis-specific events, we
established a time course of nuclear changes using Hoechst 33258. As
assessed with the Hoechst dye, control cultures showed a low level of
nuclear condensation and fragmentation (2.4 ± 1.6%) (Fig.
1C). After 2 hr of staurosporine exposure, this increased
significantly to 17.7 ± 4.3%, and by 4 hr it increased to
28.2 ± 7.2%. By 8 hr, slightly more than half of the neurons
showed a condensed or fragmented nuclear morphology. There was a
further increase up to 24 hr, the end point of observation, by which
time 76.8 ± 5.5% of the neurons were affected.
View larger version (24K):
[in this window]
[in a new window]
|
Figure 1.
Nuclear apoptotic changes in hippocampal neurons
exposed to staurosporine. A, B, Hoechst
33258 staining of nuclear chromatin. Cultured rat hippocampal neurons
were treated with 300 nM staurosporine or vehicle for 24 hr. Note the condensation and increased fluorescence of nuclei from
STS-treated cells as well as the appearance of nuclear fragmentation
(arrows). Scale bar, 20 µm. C, Time
course of nuclear apoptosis. Cultured rat hippocampal neurons were
treated with 300 nM staurosporine for the indicated periods
of time (0-24 hr). Nuclei were considered apoptotic if the nucleus
became increasingly bright and decreased in size or fragmented into
apoptotic bodies. Data are means ± SEM from n = 4-8 cultures in three separate experiments. *p < 0.05 with respect to control.
|
|
TMRE, a cationic, lipophilic dye was used for the estimation of
mitochondrial membrane potential (m) after
STS exposure. TMRE enters cells and reversibly accumulates in the
highly negatively charged mitochondrial matrix according to the Nernst
equation, so that potential can be measured dynamically; release of
TMRE after mitochondrial depolarization and its reuptake after
repolarization can be quantified. Cultures that had been treated with
300 nM staurosporine for the indicated times were loaded
with 100 nM TMRE, which remained present in the buffer
solution, and measured by digital video microscopy. The basal
fluorescence of individual cells remained stable over a period of 5 min, indicating that TMRE had fully equilibrated. Control cells had a
mean basal fluorescence of 18 ± 1 FlU. Subsequent statistical
analysis of control and STS-treated neurons revealed that the data were
not normally distributed and were heterogeneous with respect to
variance. Kruskal-Wallis ANOVA, however, revealed a significant
difference between groups. Thus the nonparametric MannWhitney test
was used for comparisons between groups, and the data are presented for
clarity in histogram form (Fig. 2). In
comparison with controls, there was no decline in TMRE fluorescence,
indicative of mitochondrial depolarization, up to 8 hr after the onset
of STS exposure, at which point more than half of the neurons showed a
condensed or fragmented nuclear morphology. On the contrary,
STS-treated cells showed a clear shift to higher fluorescence values,
which was already significant at 2 hr and increased further during the
course of STS treatment. The total pixel number of neuronal somata
remained unchanged up to 12 hr after addition of STS (334 ± 11 pixels for the 12 hr STS-treated cells vs 305 ± 10 for the
controls; p > 0.05; n = 69 and 77 cells, respectively), suggesting that cell shrinkage occurred only
later (Fig.
3A,E).
View larger version (26K):
[in this window]
[in a new window]
|
Figure 2.
Histogram representation of cellular TMRE
fluorescence during the course of staurosporine-induced apoptosis.
After the indicated lengths of exposure to 300 nM
staurosporine, hippocampal cultures were incubated with 100 nM TMRE, and fluorescence was acquired using digital video
microscopy. The x-axis represents fluorescence intervals
into which the data were grouped, whereas the y-axis
shows the percentage of cells from each treatment with fluorescence
values within a given interval. Values in the left
column represent baseline TMRE fluorescence
(Basal). Values in the right
column represent the peak fluorescence after exposure to 0.1 µM FCCP, when the dye is released from mitochondria after
depolarization ("fluorescence unquenching"). In both cases, there
is a progressive shift toward the right, corresponding to higher TMRE
fluorescence. p values from MannWhitney
U test comparisons with the corresponding controls are
shown under the treatment conditions. Data are from
n = 27-150 cells (see Table 1).
|
|
View larger version (49K):
[in this window]
[in a new window]
|
Figure 3.
Representative TMRE images from vehicle-treated
and 300 nM staurosporine-treated rat hippocampal cultures.
A, E, Phase-contrast (PC)
images. B, F, Baseline TMRE fluorescence.
Images were taken after loading cells with 100 nM TMRE.
C, G, Peak TMRE fluorescence after
treatment with 0.1 µM FCCP. The higher fluorescence of
staurosporine-treated cells is clearly visible. D,
H, Corresponding traces of the two experiments. After
loading with TMRE, baseline fluorescence was monitored for 5 min. Note
the stability of the values, indicating that the dye distribution was
fully equilibrated. At 5 min, the cultures were exposed to 0.1 µM FCCP, which depolarizes the mitochondria, resulting in
a characteristic increase in fluorescence ("unquenching") as the
dye concentration equilibrates with that outside the mitochondria. The
mitochondria of both control and staurosporine-treated cells could be
depolarized with FCCP. After 5 min of FCCP exposure, the buffer was
exchanged, and cells were allowed to recover m. Scale
bar, 20 µm.
|
|
STS-treated neurons respond to FCCP
To prove that STS-treated cells functionally respond to
pharmacological alteration of m and to
ensure that the increased TMRE fluorescence values were not a result of
TMRE release from the mitochondria through depolarization during dye
loading and consequent "unquenching" of the dye (see below), we
next examined the reaction of TMRE-loaded neurons to the protonophore
FCCP (0.1 µM). FCCP depolarizes mitochondria, releasing
TMRE into the cytoplasm, which is accompanied by a transient increase
in fluorescence ("unquenching") (Fig. 3) (Duchen and Biscoe, 1992;
Prehn et al., 1994). We first characterized the neuronal response to
various concentrations of FCCP and found that 0.1 µM was
sufficient to depolarize mitochondria from all cells, resulting in a
fluorescence increase. Concentrations higher than this (0.3 or 1.0 µM) did not lead to a greater TMRE release from
mitochondria (data not shown). Fluorescence values collected after 0.1 µM FCCP exposure (total exposure time: 5 min) showed the
same trend as the basal values: a gradual increase in mean TMRE
fluorescence, which first became significant at 2 hr and continued up
to 16 hr, the latest time point at which cells were imaged (Fig. 2),
thus indicating that the mitochondria of STS-treated neurons could
still be depolarized. Of note, unquenching values, calculated by
dividing TMRE fluorescence after FCCP exposure by the basal
fluorescence, were not significantly different between groups (Table
1), suggesting the absence of
mitochondrial depolarization during dye loading.
To exclude the possibility that damaged cells from the later time
points were washed out in preparation for imaging, and to ensure that
the cells whose TMRE fluorescence we measured were representative of
the entire population, we stained selected cultures with SYTO 16, a
live-cell nucleic acid probe, subsequent to imaging with TMRE. Cells
that had been treated with staurosporine for 16 hr and showed signs of
chromatin condensation, indicated by an increased SYTO 16 fluorescence,
still maintained m and could be depolarized
with FCCP (Fig. 4).
View larger version (63K):
[in this window]
[in a new window]
|
Figure 4.
m remains as nuclei condense.
Representative images of control (A-D) and 16 hr
staurosporine-treated (E-H) rat
hippocampal cultures. A, E,
Phase-contrast images. Neuronal damage is clearly visible in the 16 hr
staurosporine-treated culture. B, F, SYTO
16 staining. After their TMRE fluorescence was measured, cells were
returned to the culture medium and incubated with 1 µM
SYTO 16, a live-cell nucleic acid stain (30 min, 37°C). Nuclei of
staurosporine-treated cells show increased fluorescence, as well as a
more condensed and irregular morphology. C,
G, Baseline TMRE fluorescence. Cells that show evidence
of nuclear condensation in the SYTO 16 image (arrows)
maintain m. TMRE fluorescence of the
staurosporine-treated cells is actually higher than that of controls.
D, H, Peak TMRE fluorescence after 0.1 µM FCCP treatment. Here the difference between the
control and treated cells is even more pronounced. Cells that show
signs of nuclear apoptosis can still be depolarized by FCCP. Scale bar,
20 µm.
|
|
Mitochondrial mass remains stabile during STS-induced
neuronal apoptosis
Increases in both basal and FCCP-induced TMRE fluorescence could
indicate an increase in m, mitochondrial
volume, mitochondrial mass, or even plasma membrane potential. In an
attempt to differentiate between these possibilities, we first used the
probe Mitotracker Green FM, an indicator of mitochondrial mass
(Metivier et al., 1998). A preliminary experiment in hippocampal
neurons showed that uptake of this dye, in contrast to TMRE, was indeed
not dependent on m (Table
2). Additional studies performed in PC12
pheochromocytoma cells indicated that uptake of this probe was also not
influenced by exposure to FCCP (10 µM, 30 min),
oligomycin (5 µg/ml, 30 min), the Ca2+
ionophore A-23187 in combination with thapsigargin (5 and 1 µM, 4 hr), or mastoparan (50 µM, 4 hr)
(data not shown). In subsequent experiments, we found that Mitotracker
Green FM fluorescence of the hippocampal neuron cultures remained
unchanged up to 12 hr after the start of staurosporine exposure (Fig.
5A). In addition, we found
that uptake of another probe used to determine changes in mitochondrial
mass, nonyl acridine orange (Cossarizza et al., 1995; Mancini et al.,
1997; Metivier et al., 1998), also remained unchanged up to 12 hr after
STS exposure (Fig. 5B).
View larger version (19K):
[in this window]
[in a new window]
|
Figure 5.
Measurement of mitochondrial mass using
Mitotracker Green FM (MTGFM) and nonyl acridine orange
(NAO). Cultured rat hippocampal neurons exposed to STS
for the indicated times were loaded with MTGFM (200 nM, 30 min) or NAO (1 µg/ml) in medium for 30 min at 37°C, and the
fluorescence of cell extracts was measured using a fluorescence plate
reader. No significant differences were found with respect to the
controls. Data are the means ± SEM from n = 6 cultures. The experiments were repeated with similar results.
|
|
Incubation in high K+ buffer abolishes the
STS-induced increase in TMRE fluorescence
Because mitochondrial mass remained unchanged during
STS-induced apoptosis, we next examined the possibility that an
increase in plasma membrane potential was in fact responsible for the
increased uptake of TMRE after STS treatment. The Nernstian behavior of this cationic probe would predict a concomitant increase in uptake as
either mitochondrial or plasma potential increases (Ehrenberg et al.,
1988). Plasma membrane hyperpolarization attributable to enhancement of
outward potassium current has been observed in several models of
neuronal apoptosis (Yu et al., 1997). To eliminate this influence, a
high K+ buffer containing 50 mM KCl was used to depolarize the plasma membrane.
Hippocampal neurons exposed to 300 nM STS for 6 hr and loaded with TMRE again showed an increase in fluorescence compared with
controls (Fig. 6). In contrast,
incubation in high K+ buffer decreased
mitochondrial TMRE uptake in control cells but also prevented the
increase in TMRE uptake seen after STS treatment. A reduction of the
STS-induced increase in mitochondrial TMRE uptake was also observed in
cultures treated with the potassium channel blockers tetraethylammonium
(25 mM) and clofilium (1 µM) 15 min before
TMRE loading (data not shown).
View larger version (12K):
[in this window]
[in a new window]
|
Figure 6.
The STS-induced increase in cellular TMRE
fluorescence is abolished by incubation in high K+
buffer. Hippocampal neurons were treated with 300 nM STS
for 6 hr. After this, the medium was exchanged for
Ca2+-free HBS containing either 5 mM
(Control) or 50 mM (High
K+) KCl, in which the cells were incubated for 5 min before the addition of 100 nM TMRE. After a 10 min
loading period, cellular TMRE fluorescence was acquired. Data are
presented in median/quartile form and represent values from
n = 100-137 cells in two experiments per
treatment. *p < 0.05 with respect to
control.
|
|
Cytochrome c release proceeds without loss
of m
Because we found no evidence of early mitochondrial
depolarization in our experiments with TMRE, we next examined one
possible consequence of mitochondrial depolarization-release of
cytochrome c, which has been implicated in apoptotic cell death-with
the aid of immunofluorescence microscopy in con- junction with
monitoring of m using the
potential-sensitive probe Mitotracker Red CMXRos. Unlike TMRE, CMXRos
forms stable thiol conjugates with cellular peptides, inhibiting
release of the dye from mitochondria after loading. In addition, the
formation of thiol conjugates allows fixation and double-labeling of
cells with an antibody to cytochrome c. Comparable to the results seen
with TMRE, we saw no decrease in neuronal CMXRos fluorescence after
addition of STS but rather an increase by 8 hr (Fig.
7A,C).
In contrast, cytochrome c staining of STS-treated cells was clearly
lower than that of the respective controls by this time (Fig.
7B,D). In fact, cells that had
already lost a considerable amount of cytochrome c by 8 hr exhibited
increased CMXRos fluorescence (Fig. 7, arrows). Furthermore,
in many cells we observed a transition from a punctate to a diffuse
staining pattern. Our observation of a decrease in cytochrome c
staining could be indicative of cytochrome c modification or
degradation after its release into the cytoplasm (Neame et al., 1998;
Varkey et al., 1999).
View larger version (72K):
[in this window]
[in a new window]
|
Figure 7.
Cytochrome c is released while
m remains stabile. Cultured rat hippocampal neurons
were exposed to either vehicle (A, B) or
300 nM STS (C, D) for 8 hr, then incubated
with 50 nM CMXRos for 30 min at 37°C. After fixation and
permeabilization, cultures were subjected to immunocytochemistry using
an antibody specific to native cytochrome c in conjunction with
biotinylated anti-mouse IgG and streptavidin-conjugated Oregon Green.
A, C, CMXRos staining to visualize
changes in m. Note that in the majority of neurons,
CMXRos fluorescence, indicative of m,
increases during STS exposure. B, D,
Cytochrome c immunofluorescence observed with Oregon Green. In contrast
to that seen with CMXRos, Oregon Green fluorescence decreases
considerably during STS exposure. Arrows in
C and D indicate cells with particularly
pronounced increases in CMXRos fluorescence and decreases in Oregon
Green fluorescence compared with controls. Scale bar, 20 µm.
|
|
A similar pattern was seen when the cultures were examined with a 100×
oil objective, allowing resolution of individual mitochrondria and
mitochondria-rich regions (Fig. 8).
Mitochondria from STS-treated cells showed only a small level of
staining for cytochrome c compared with controls; this was even the
case in mitochondria and mitochondria-rich regions that showed a high
level of staining with CMXRos (Fig. 8B,E).
View larger version (24K):
[in this window]
[in a new window]
|
Figure 8.
High-resolution imaging of cytochrome c release.
Control (A-C) and STS-treated
(D-F) hippocampal cultures were treated as in
Figure 7. CMXRos (m) and Oregon Green
(cytochrome c) staining were then observed with a 100× oil objective.
A, D, CMXRos staining for
m. Mitochondria from STS-treated cells show no loss
of m 8 hr after addition of the drug.
B, E, Cytochrome c immunofluorescence
staining with Oregon Green. Mitochondria from control cells show a high
level of staining for cytochrome c. In comparison, mitochondria from
STS-treated cells show extensive loss of cytochrome c staining
encompassing most of the mitochondrial population. C,
F, Superimposed images of CMXRos (red)
and Oregon Green staining. Yellow areas are indicative
of colocalized CMXRos and Oregon Green staining. Arrows
indicate areas with especially pronounced loss of staining for
cytochrome c without a corresponding decrease in m.
Scale bar, 5 µm.
|
|
Time course of caspase-3-like activity
Cytochrome c release from mitochondria can lead to activation of
the caspase cascade through consecutive activation of caspase-9 and
caspase-3 (Li et al., 1997). To assess whether caspase activation could
proceed without mitochondrial depolarization, we measured caspase-3-like activity by monitoring the cleavage of a fluorogenic caspase substrate by extracts from staurosporine-treated neurons. Ac-DEVD-AMC is cleaved efficiently by caspase-3 but also by caspase-1, -4, -6, -7, and -8 (Talanian et al., 1997; Garcia-Calvo et al., 1999).
Production of the fluorescent cleavage product AMC was negligible with
extracts from control cultures (1.1 ± 0.6 FlU · 120
min 1 · µg1 protein) (Fig.
9). By 2 hr of staurosporine exposure,
cleavage activity had increased significantly to 7.6 ± 1.1 FlU · 120
min1 · µg1 protein. By 8 hr, at which
point there had been no decrease in m,
caspase-3-like activity reached a maximum, 23.6 ± 0.7 FlU · 120
min1 · µg1 protein, a >20-fold
increase compared with the control. Cleavage activity then decreased by
approximately one-half between 8 and 12 hr. Overall protein content
remained unchanged up to 12 hr after addition of STS (data not shown).
We also obtained evidence for the activation of caspases by Western
blotting of caspase substrates. We observed cleavage of both
poly-(ADP-ribose) polymerase, which is cleaved most efficiently by
caspase-3 but also by other caspases (Margolin et al., 1997), as well
as  -spectrin, both reaching a maximum by 6 hr after STS addition
(data not shown). Thus, cytochrome c release was accompanied by
activation of caspase-3-like proteases, without a corresponding
decrease in m.
View larger version (14K):
[in this window]
[in a new window]
|
Figure 9.
Time course of caspase-3-like protease activity in
cytosolic protein extracts. Cultured rat hippocampal neurons were
treated with staurosporine (300 nM) for the indicated
periods of time. Caspase-3-like protease activity was measured by
cleavage of the fluorogenic substrate Ac-DEVD-AMC (10 µM). Activities are represented as increase in AMC
fluorescence [in arbitrary fluorescence units (Fl.U.)]
over 120 min per microgram protein. Data are means ± SEM
from n = 6 cultures. A duplicate experiment yielded
similar results. Different from controls, *p < 0.05.
|
|
No prevention of staurosporine-induced neuronal death by
cyclosporine A or bongkrekic acid, inhibitors of the mitochondrial
permeability transition
We pretreated hippocampal cultures with cyclosporine A (10 µM) or bongkrekic acid (50 µM), two potent
inhibitors of PTP with distinct modes of action, 1 hr before exposure
to 300 nM staurosporine. The percentage of apoptotic nuclei
was assayed 24 hr after the start of staurosporine exposure by Hoechst
33258 staining. Cyclosporine A and bongkrekic acid both showed no
significant toxicity when added alone (Fig.
10). Neither PTP inhibitor reduced the
level of apoptotic nuclear morphology after STS exposure. Cell
viability, assessed by trypan blue staining, was also unaffected (data
not shown). In addition, loss of mitochondrial cytochrome c was not influenced by treatment with cyclosporine A (45.3 ± 2.5% neurons showing loss of mitochondrial cytochrome c immunofluorescence 6 hr
after STS treatment vs 52.5 ± 6.4% in STS-treated and
cyclosporine A-treated cultures; n = 4 cultures per
treatment; p > 0.1).
View larger version (13K):
[in this window]
[in a new window]
|
Figure 10.
Inhibitors of the mitochondrial PTP do not
prevent STS-induced apoptosis. Hippocampal neurons were pretreated for
1 hr with cyclosporine A (CsA, 10 µM) or
bongkrekic acid (BA, 50 µM), then 300 nM STS was added to the culture medium. After 24 hr of STS
treatment, apoptotic cell death was assessed by Hoechst 33258 staining
and determined as in Figure 1. Data are means ± SEM from
n = 4 cultures. *p < 0.05 with
respect to control. A duplicate experiment yielded comparable
results.
|
|
| |
DISCUSSION |
In an attempt to clarify the importance of mitochondrial membrane
potential in PCD of rat hippocampal neurons, we investigated the time
courses of m, cytochrome c release, caspase
activation, and apoptotic nuclear changes during staurosporine-induced
apoptosis of cultured rat hippocampal neurons. Treatment with 300 nM staurosporine led to rapid, apoptotic cell death, which
was characterized by the appearance of nuclear apoptotic morphology
visualized with the chromatin stain Hoechst 33258. The percentage of
nuclei showing condensation and fragmentation was already significantly
higher than the controls by 2 hr after staurosporine treatment and by 8 hr had increased to more than half the cell population (Fig. 1C). Caspase-3-like activity increased significantly (by
nearly sevenfold) as early as 2 hr after staurosporine exposure and
peaked 8 hr after the start of treatment (Fig. 9), at which point
mitochondrial release of cytochrome c, an inducer of caspase activity,
was readily apparent (Figs. 7, 8).
In this context it is especially interesting that we observed no
mitochondrial depolarization during the course of these
staurosporine-induced apoptotic changes using either TMRE or CMXRos,
probes that accumulate reversibly and irreversibly, respectively, in
mitochondria according to the magnitude of
m. Particularly important, cells could be depolarized with FCCP throughout the length of staurosporine treatment, as shown with the reversible probe TMRE. Interestingly, even individual cells that were in the later stages of apoptosis (showing apoptotic nuclear changes as seen with SYTO 16) could be shown to maintain mitochondrial function (Fig. 4).
We cannot fully discount the possibility that only a small portion of
mitochondria depolarized, which might be sufficient to trigger events
downstream. In our high-magnification observations with the probe
CMXRos, however, we found no such evidence at the subcellular level
(Fig. 8). It is indeed conceivable that such a strategy would be risky
for the organism, because a small number of mitochondria could
determine the fate of entire cells (Murphy et al., 1999). Our results
are in agreement with the thought that apoptosis is an active process
during which cells must maintain sufficient ATP levels and thus,
presumably, mitochondrial function (Nicotera and Leist, 1997).
We also examined the possibility that the mitochondrial PTP might be
involved in staurosporine-induced PCD. Evidence for involvement of PTP
in apoptosis includes the observation that known inhibitors of PTP such
as cyclosporine A and bongkrekic acid prevent a number of
apoptosis-associated events (Marchetti et al., 1996; Pastorino et al.,
1996; Zamzami et al., 1996b; Marzo et al., 1998). A 1 hr pretreatment
with cyclosporine A or bongkrekic acid (10 and 50 µM,
respectively) failed to prevent apoptosis after subsequent treatment
with staurosporine (Fig. 10), suggesting that PTP is not necessary for
the initiation of STS-induced apoptosis. The TMRE fluorescence data are
further evidence against the participation of a PTP in this model. The
opening of a PTP leads to loss of m,
because the mitochondrion is no longer able to maintain a proton
gradient across the inner membrane (Zoratti and Szabo, 1994; Zamzami et
al., 1996a). We observed no such loss of m during STS-induced apoptosis.
By 8 hr, when more than one-half the cell population showed signs of
nuclear apoptosis, cytochrome c release was evident, and caspase-3-like
activity had peaked, TMRE baseline fluorescence had actually increased
by roughly 30% (Fig. 2). A similar increase in CMXRos fluorescence was
also seen at this time point. By 16 hr after treatment, the increase in
TMRE fluorescence was roughly 70%. This increase in TMRE fluorescence
seen during the course of neuronal apoptosis could be caused by
mitochondrial hyperpolarization/swelling (Vander Heiden et al., 1997),
an increase in mitochondrial mass (Mancini et al., 1997), or plasma
membrane hyperpolarization (Ehrenberg et al., 1988). Our results
support the latter alternative. We examined cellular uptake of
Mitotracker Green FM and nonyl acridine orange, two probes used to
evaluate changes in mitochondrial mass, and found no change in
fluorescence up to 12 hr after the start of staurosporine treatment
(Fig. 5). On the other hand, incubation of cells in high
K+ buffer was sufficient to prevent the
increase in TMRE uptake seen after STS treatment (Fig. 6), suggesting
that this increase was caused at least partly by an increase in plasma
membrane potential. Interestingly, Yu et al. (1997) found an increase
in outward K+ current during neuronal
apoptosis induced by STS or serum deprivation. Blocking this increase
with high K+ or tetraethylammonium reduced
the level of apoptosis, which suggests the potential importance of the
plasma membrane potential in apoptotic cell death processes.
On the other hand, there is support for the idea that mitochondrial
volume homeostasis could be involved in apoptosis and that
anti-apoptotic Bcl-2-family proteins might function by stabilizing m and preventing swelling [Vander Heiden
et al. (1997, 1999); but see Shimizu et al. (1998)]. It has been
suggested that mitochondrial swelling could result in rupture of the
outer membrane, a possible mechanism of release of pro-apoptotic
factors (Vander Heiden et al., 1997). Our observations of neurons
incubated in high K+ buffer
notwithstanding, it is possible that the increase in TMRE and CMXRos
fluorescence that we observed could reflect an actual increase in
m or mitochondrial volume secondary to a
defect in mitochondrial ADP/ATP exchange (Vander Heiden et al., 1999). Moreover, mitochondrial hyperpolarization may increase the likelihood of mitochondrial generation of superoxide (Korshunov et al., 1997), an
event that significantly contributes to STS-induced neuronal cell death
(Krohn et al., 1998). Alternatively, the release of pro-apoptotic
factors could be controlled by specific channels or contact points
located in the mitochondrial membrane (Reed, 1997b), could be subject
to specific proteolytic processes, or could be caused by a reversal of
protein import processes.
Although it is becoming clearer that mitochondria play an important
role in PCD, especially in light of evidence that the release of
pro-apoptotic factors from the mitochondria can initiate the caspase
cascade, there is much conflicting data concerning the role of
mitochondria upstream of this event. Using STS-induced cell death in
cultured rat hippocampal neurons, we demonstrate that cytochrome c
release, caspase activation, and nuclear apoptosis may proceed
independently of loss of mitochondrial potential. We also show that
mitochondria of neurons displaying morphological signs of apoptosis
functionally respond to a depolarizing agent. Taken together, our data
suggest that mitochondrial depolarization is not required for
STS-induced neuronal apoptosis. This model of neuronal cell death may
have broad biological significance, because it involves inhibition of
survival kinases, increased oxidant stress, and a disturbance of
neuronal Ca2+ homeostasis (Bertrand et
al., 1994; Prehn et al., 1997; Krohn et al., 1998; Kumar and Mattson,
1999), findings that have been suggested to be involved in neuronal
cell death in neurodegenerative disorders.
| |
FOOTNOTES |
Received Dec. 30, 1998; revised May 14, 1999; accepted June 10, 1999.
This work was supported by a grant from Alzheimer Forschung Initiative
e.V. to J.H.M.P. and IZKF Universität Münster. We thank Elke Bauerbach for technical assistance and Professor J. Krieglstein for his support.
Correspondence should be addressed to Dr. Jochen H. M. Prehn,
Interdisciplinary Center for Clinical Research (IZKF), Junior Research
Group "Apoptosis and Cell Death," Faculty of Medicine, Westphalian
Wilhelms-University, Röntgenstrasse 21, D-48149 Münster, Germany.
| |
REFERENCES |
-
Ankarcrona M,
Dypbukt JM,
Bonfoco E,
Zhivotovsky B,
Orrenius S,
Lipton SA,
Nicotera P
(1995)
Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function.
Neuron
15:961-973[Web of Science][Medline].
-
Bertrand R,
Solary E,
O'Connor P,
Kohn KW,
Pommier Y
(1994)
Induction of a common pathway of apoptosis by staurosporine.
Exp Cell Res
211:314-321[Web of Science][Medline].
-
Boise LH,
Thompson CB
(1997)
Bcl-xl can inhibit apoptosis in cells that have undergone Fas-induced protease activation.
Proc Natl Acad Sci USA
94:3759-3764[Abstract/Free Full Text].
-
Bossy-Wetzel E,
Newmeyer DD,
Green DR
(1998)
Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization.
EMBO J
17:37-49[Web of Science][Medline].
-
Cohen GM
(1997)
Caspases: the executioners of apoptosis.
Biochem J
326:1-16.
-
Cossarizza A,
Cooper EL,
Quaglino D,
Salvioli S,
Kalachnikova G,
Franceschi C
(1995)
Mitochondrial mass and membrane potential in coelomocytes from the earthworm Eisenia foetida: studies with fluorescent probes in single intact cells.
Biochem Biophys Res Commun
214:503-510[Web of Science][Medline].
-
Duchen MR,
Biscoe TJ
(1992)
Relative mitochondrial membrane potential and [Ca2+] in type I cells isolated from the rabbit carotid body.
J Physiol (Lond)
450:33-61[Abstract/Free Full Text].
-
Ehrenberg B,
Montana V,
Wie MD,
Wuskell JP,
Loew LM
(1988)
Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes.
Biophys J
53:785-794[Web of Science][Medline].
-
Falcieri E,
Martelli AM,
Bareggi R,
Cataldi A,
Cocco L
(1993)
The protein kinase inhibitor staurosporine induces morphological changes typical of apoptosis in MOLT-4 cells without concomitant DNA fragmentation.
Biochem Biophys Res Commun
193:19-25[Web of Science][Medline].
-
Frey T
(1995)
Nucleic acid dyes for detection of apoptosis in live cells.
Cytometry
21:265-274[Web of Science][Medline].
-
Garcia-Calvo M,
Peterson EP,
Rasper DM,
Vaillancourt JP,
Zamboni R,
Nicholson DW,
Thornberry NA
(1999)
Purification and catalytic properties of human caspase family members.
Cell Death Differ
6:362-369.[Web of Science][Medline]
-
Gunter TE,
Pfeiffer DR
(1990)
Mechanisms by which mitochondria transport calcium.
Am J Physiol
258:C755-C786[Abstract/Free Full Text].
-
Heiskanen KM,
Bhat MB,
Wang H-W,
Ma J,
Nieminen A-L
(1999)
Mitochondrial depolarization accompanies cytochrome c release during apoptosis in PC6 cells.
J Biol Chem
274:5654-5658[Abstract/Free Full Text].
-
Kluck RM,
Bossy-Wetzel E,
Green DR,
Newmeyer DD
(1997)
The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis.
Science
275:1132-1136[Abstract/Free Full Text].
-
Koh J-Y,
Wie MB,
Gwag BJ,
Sensi SL,
Canzoniero LMT,
Demaro J,
Csernansky C,
Choi DW
(1995)
Staurosporine-induced neuronal apoptosis.
Exp Neurol
135:153-159[Web of Science][Medline].
-
Korshunov SS,
Skulachev VP,
Starkov AA
(1997)
High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria.
FEBS Lett
416:15-18[Web of Science][Medline].
-
Kroemer G,
Zamzami N,
Susin SA
(1997)
Mitochondrial control of apoptosis.
Immun Today
18:44-51.
-
Krohn AJ,
Preis E,
Prehn JHM
(1998)
Staurosporine-induced apoptosis of cultured rat hippocampal neurons involves caspase-1-like proteases as upstream initiators and increased production of superoxide as a main downstream effector.
J Neurosci
18:8186-8197[Abstract/Free Full Text].
-
Kumar I,
Mattson MP
(1999)
Pivotal role of mitochondrial calcium uptake in neural cell apoptosis and necrosis.
J Neurochem
72:529-540[Web of Science][Medline].
-
Li P,
Nijhawan D,
Budihardjo I,
Srinivasula SM,
Ahmad M,
Alnemri ES,
Wang X
(1997)
Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade.
Cell
91:479-489[Web of Science][Medline].
-
Mancini M,
Anderson BO,
Caldwell E,
Sedghinasab M,
Paty PB,
Hockenbery DM
(1997)
Mitochondrial proliferation and paradoxical membrane depolarization during terminal differentiation and apoptosis in a human colon carcinoma cell line.
J Cell Biol
138:449-469[Abstract/Free Full Text].
-
Marchetti P,
Castedo M,
Susin SA,
Zamzami N,
Hirsch T,
Macho A,
Haeffner A,
Hirsch F,
Geuskens M,
Kroemer G
(1996)
Mitochondrial permeability transition is a central coordinating event of apoptosis.
J Exp Med
184:1155-1160[Abstract/Free Full Text].
-
Margolin N,
Raybuck SA,
Wilson KP,
Chen W,
Fox T,
Gu Y,
Livingston DJ
(1997)
Substrate and inhibitor specificity of interleukin-1-converting enzyme and related caspases.
J Biol Chem
272:7223-7228[Abstract/Free Full Text].
-
Marzo I,
Brenner C,
Zamzami N,
Jürgensmeier JM,
Susin SA,
Vieira HLA,
Prévost M-C,
Xie Y,
Matsuyama S,
Reed JC,
Kroemer G
(1998)
Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis.
Science
281:2027-2031[Abstract/Free Full Text].
-
Metivier D,
Dallaporta B,
Zamzami N,
Larochette N,
Susin SA,
Marzo I,
Kroemer G
(1998)
Cytofluorometric detection of mitochondrial alterations in early CD95/Fas/APO-1-triggered apoptosis of Jurkat T lymphoma cells: comparison of seven mitochondrion-specific fluorochromes.
Immunol Lett
61:157-163[Web of Science][Medline].
-
Monck JR,
Oberhauser AF,
Keating TJ,
Fernandez JM
(1992)
Thin-section ratiometric Ca2+ images obtained by optical sectioning of fura-2 loaded mast cells.
J Cell Biol
116:745-759[Abstract/Free Full Text].
-
Murphy AN,
Fiskum G,
Flint Beal M
(1999)
Mitochondria in neurodegeneration: bioenergetic function in cell life and death.
J Cereb Bood Flow Metab
19:231-245.
-
Neame SJ,
Rubin LL,
Philpott KL
(1998)
Blocking cytochrome c activity within intact neurons inhibits apoptosis.
J Cell Biol
142:1583-1593[Abstract/Free Full Text].
-
Nicotera P,
Leist M
(1997)
Energy supply and the shape of cell death in neurons and lymphoid cells.
Cell Death Differ
4:435-442.
-
Pastorino JG,
Simbula G,
Yamamoto K,
Glascott Jr PA,
Rothman RL,
Farber JL
(1996)
The cytotoxicity of tumor necrosis factor depends on induction of the mitochondrial permeability transition.
J Biol Chem
271:29792-29798[Abstract/Free Full Text].
-
Prehn JHM
(1998)
Mitochondrial transmembrane potential and free radical production in excitotoxic neurodegeneration.
Naunyn Schmiedebergs Arch Pharmacol
357:316-322[Web of Science][Medline].
-
Prehn JHM,
Bindokas VP,
Marcuccilli CJ,
Krajewski S,
Reed JC,
Miller RJ
(1994)
Regulation of neuronal Bcl2 protein expression and calcium homeostasis by transforming growth factor type confers wide-ranging protection on rat hippocampal neurons.
Proc Natl Acad Sci USA
91:12599-12603[Abstract/Free Full Text].
-
Prehn JHM,
Jordan J,
Ghadge GD,
Preis E,
Galindo MF,
Roos RP,
Krieglstein J,
Miller RJ
(1997)
Ca2+ and reactive oxygen species in staurosporine-induced neuronal apoptosis.
J Neurochem
68:1679-1685[Web of Science][Medline].
-
Reed JC
(1997a)
Double identity for proteins of the Bcl-2 family.
Nature
387:773-776[Medline].
-
Reed JC
(1997b)
Cytochrome c: can't live with it-can't live without it.
Cell
91:559-562[Web of Science][Medline].
-
Salvesen GS,
Dixit VM
(1997)
Caspases: intracellular signaling by proteolysis.
Cell
91:443-446[Web of Science][Medline].
-
Shimizu S,
Eguchi Y,
Kamiike W,
Funahashi Y,
Mignon A,
Lacronique V,
Matsuda H,
Tsujimoto Y
(1998)
Bcl-2 prevents apoptotic mitochondrial dysfunction by regulating proton flux.
Proc Natl Acad Sci USA
95:1455-1459[Abstract/Free Full Text].
-
Susin SA,
Zamzami N,
Castedo M,
Hirsch T,
Marchetti P,
Macho A,
Daugas E,
Geuskens M,
Kroemer G
(1996)
Bcl-2 inhibits the mitochondrial release of an apoptogenic protease.
J Exp Med
184:1331-1342[Abstract/Free Full Text].
-
Susin SA,
Lorenzo HK,
Zamzami N,
Marzo I,
Snow BE,
Brothers GM,
Mangion J,
Jacotot E,
Costantini P,
Loeffler M,
Larochette N,
Goodlet DR,
Aebersold R,
Siderovski DP,
Penninger JM,
Kroemer G
(1999)
Molecular characterization of mitochondrial apoptosis-inducing factor.
Nature
397:441-446[Medline].
-
Talanian RV,
Quinlan C,
Trautz S,
Hackett MC,
Mankovich JA,
Banach D,
Ghayur T,
Brady KD,
Wong WW
(1997)
Substrate specificity of caspase family proteases.
J Biol Chem
272:9677-9682[Abstract/Free Full Text].
-
Thompson CB
(1995)
Apoptosis in the pathogenesis and treatment of disease.
Science
267:1456-1462[Abstract/Free Full Text].
-
Tsujimoto Y
(1997)
Apoptosis and necrosis: intracellular ATP level as a determinant for cell death modes.
Cell Death Differ
4:429-434.
-
Vander Heiden MG,
Chandel NS,
Williamson EK,
Schumacker PT,
Thompson CB
(1997)
Bcl-xl regulates the membrane potential and volume homeostasis of mitochondria.
Cell
91:627-637[Web of Science][Medline].
-
Vander Heiden MG,
Chandel NS,
Schumacker PT,
Thompson CB
(1999)
Bcl-xl prevents cell death following growth factor withdrawal by facilitating mitochondrial ATP/ADP exchange.
Mol Cell
3:159-167.[Web of Science][Medline]
-
Varkey J,
Chen P,
Jemmerson R,
Abrams JM
(1999)
Altered cytochrome c display precedes apoptotic cell death in Drosophila.
J Cell Biol
144:701-710[Abstract/Free Full Text].
-
Wadia JS,
Chalmers-Redman RME,
Ju WJH,
Carlile GW,
Phillips JL,
Fraser AD,
Tatton WG
(1998)
Mitochondrial membrane potential and nuclear changes in apoptosis caused by serum and nerve growth factor withdrawal: time course and modification by ()-deprenyl.
J Neurosci
18:932-947[Abstract/Free Full Text].
-
Yang J,
Liu X,
Bhalla K,
Kim CN,
Ibrado AM,
Cai J,
Peng TI,
Jones DP,
Wang X
(1997)
Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.
Science
275:1129-1132[Abstract/Free Full Text].
-
Yoshida H,
Kong YY,
Yoshida R,
Elia AJ,
Hakem A,
Hakem R,
Penninger JM,
Mak TW
(1998)
Apaf1 is required for mitochondrial pathways of apoptosis and brain development.
Cell
94:739-750[Web of Science][Medline].
-
Yu SP,
Yeh CH,
Sensi SL,
Gwag BJ,
Canzoniero LMT,
Farhangrazi ZS,
Ying HS,
Tian M,
Dugan LL,
Choi DW
(1997)
Mediation of neuronal apoptosis by enhancement of outward potassium current.
Science
278:114-118[Abstract/Free Full Text].
-
Zamzami N,
Susin SA,
Marchetti P,
Hirsch T,
Gomez-Monterrey I,
Castedo M,
Kroemer G
(1996a)
Mitochondrial control of nuclear apoptosis.
J Exp Med
183:1533-1544[Abstract/Free Full Text].
-
Zamzami N,
Marchetti P,
Castedo M,
Hirsch T,
Susin SA,
Masse B,
Kroemer G
(1996b)
Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis.
FEBS Lett
384:53-57[Web of Science][Medline].
-
Zoratti M,
Szabo I
(1994)
Electrophysiology of the inner mitochondrial membrane.
J Bioenerg Biomembr
26:543-553[Web of Science][Medline].
-
Zoratti M,
Szabo I
(1995)
The mitochondrial permeability transition.
Biochim Biophys Acta
1241:139-176[Medline].
-
Zou H,
Henzel WJ,
Liu X,
Lutschg A,
Wang X
(1997)
Apaf-1, a human protein homologous to C. elegans CED-4: participates in cytochrome c-dependent activation of caspase-3.
Cell
90:405-413[Web of Science][Medline].
Copyright © 1999 Society for Neuroscience 0270-6474/99/19177394-11$05.00/0
This article has been cited by other articles:
|
|
|
|
 
J. P. Norman, S. W. Perry, K. A. Kasischke, D. J. Volsky, and H. A. Gelbard
HIV-1 Trans Activator of Transcription Protein Elicits Mitochondrial Hyperpolarization and Respiratory Deficit, with Dysregulation of Complex IV and Nicotinamide Adenine Dinucleotide Homeostasis in Cortical Neurons
J. Immunol.,
January 15, 2007;
178(2):
869 - 876.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Zhu, Y. Lai, P. B. Shelat, C. Hu, G. Y. Sun, and J. C-M. Lee
Phospholipases A2 Mediate Amyloid-beta Peptide-Induced Mitochondrial Dysfunction
J. Neurosci.,
October 25, 2006;
26(43):
11111 - 11119.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. C. David, S. Hauptmann, I. Scherping, K. Schuessel, U. Keil, P. Rizzu, R. Ravid, S. Drose, U. Brandt, W. E. Muller, et al.
Proteomic and Functional Analyses Reveal a Mitochondrial Dysfunction in P301L Tau Transgenic Mice
J. Biol. Chem.,
June 24, 2005;
280(25):
23802 - 23814.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. W. Perry, J. P. Norman, A. Litzburg, D. Zhang, S. Dewhurst, and H. A. Gelbard
HIV-1 Transactivator of Transcription Protein Induces Mitochondrial Hyperpolarization and Synaptic Stress Leading to Apoptosis
J. Immunol.,
April 1, 2005;
174(7):
4333 - 4344.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Dong, Q. Li, S.-c. Lyu, A. M. Krensky, and C. Clayberger
A novel apoptosis pathway activated by the carboxyl terminus of p21
Blood,
February 1, 2005;
105(3):
1187 - 1194.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
U. Keil, A. Bonert, C. A. Marques, I. Scherping, J. Weyermann, J. B. Strosznajder, F. Muller-Spahn, C. Haass, C. Czech, L. Pradier, et al.
Amyloid {beta}-induced Changes in Nitric Oxide Production and Mitochondrial Activity Lead to Apoptosis
J. Biol. Chem.,
November 26, 2004;
279(48):
50310 - 50320.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Tezel and X. Yang
Caspase-Independent Component of Retinal Ganglion Cell Death, In Vitro
Invest. Ophthalmol. Vis. Sci.,
November 1, 2004;
45(11):
4049 - 4059.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. L. Galindo, A. A. Fadl, J. Sha, C. Gutierrez Jr., V. L. Popov, I. Boldogh, B. B. Aggarwal, and A. K. Chopra
Aeromonas hydrophila Cytotoxic Enterotoxin Activates Mitogen-activated Protein Kinases and Induces Apoptosis in Murine Macrophages and Human Intestinal Epithelial Cells
J. Biol. Chem.,
September 3, 2004;
279(36):
37597 - 37612.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. P. Vrabec, C. J. Lieven, and L. A. Levin
Cell-Type-Specific Opening of the Retinal Ganglion Cell Mitochondrial Permeability Transition Pore
Invest. Ophthalmol. Vis. Sci.,
June 1, 2003;
44(6):
2774 - 2782.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Q. Wang, A. Y. Xiao, C. Sheline, K. Hyrc, A. Yang, M. P. Goldberg, D. W. Choi, and S. Ping Yu
Apoptotic insults impair Na+, K+-ATPase activity as a mechanism of neuronal death mediated by concurrent ATP deficiency and oxidant stress
J. Cell Sci.,
May 15, 2003;
116(10):
2099 - 2110.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Dussmann, M. Rehm, D. Kogel, and J. H. M. Prehn
Outer mitochondrial membrane permeabilization during apoptosis triggers caspase-independent mitochondrial and caspase-dependent plasma membrane potential depolarization: a single-cell analysis
J. Cell Sci.,
February 1, 2003;
116(3):
525 - 536.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. A. Linseman, R. A. Phelps, R. J. Bouchard, S. S. Le, T. A. Laessig, M. L. McClure, and K. A. Heidenreich
Insulin-Like Growth Factor-I Blocks Bcl-2 Interacting Mediator of Cell Death (Bim) Induction and Intrinsic Death Signaling in Cerebellar Granule Neurons
J. Neurosci.,
November 1, 2002;
22(21):
9287 - 9297.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S.-G. Huang
Development of a High Throughput Screening Assay for Mitochondrial Membrane Potential in Living Cells
J Biomol Screen,
August 1, 2002;
7(4):
383 - 389.
[Abstract]
[PDF]
|
|
|
|
|
|
|
Q. Yuan, R. M. Ray, and L. R. Johnson
Polyamine depletion prevents camptothecin-induced apoptosis by inhibiting the release of cytochrome c
Am J Physiol Cell Physiol,
June 1, 2002;
282(6):
C1290 - C1297.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Wobser, H. Du{beta}mann, D. Kogel, H. Wang, C. Reimertz, C. B. Wollheim, M. M. Byrne, and J. H. M. Prehn
Dominant-negative Suppression of HNF-1alpha Results in Mitochondrial Dysfunction, INS-1 Cell Apoptosis, and Increased Sensitivity to Ceramide-, but Not to High Glucose-induced Cell Death
J. Biol. Chem.,
February 15, 2002;
277(8):
6413 - 6421.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Krick, O. Platoshyn, M. Sweeney, S. S. McDaniel, S. Zhang, L. J. Rubin, and J. X.-J. Yuan
Nitric oxide induces apoptosis by activating K+ channels in pulmonary vascular smooth muscle cells
Am J Physiol Heart Circ Physiol,
January 1, 2002;
282(1):
H184 - H193.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. A. Reichert, J. S. Kim-Han, and L. L. Dugan
The Mitochondrial Permeability Transition Pore and Nitric Oxide Synthase Mediate Early Mitochondrial Depolarization in Astrocytes during Oxygen-Glucose Deprivation
J. Neurosci.,
September 1, 2001;
21(17):
6608 - 6616.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Poppe, C. Reimertz, H. Du{beta}mann, A. J. Krohn, C. M. Luetjens, D. Bockelmann, A.-L. Nieminen, D. Kogel, and J. H. M. Prehn
Dissipation of Potassium and Proton Gradients Inhibits Mitochondrial Hyperpolarization and Cytochrome c Release during Neural Apoptosis
J. Neurosci.,
July 1, 2001;
21(13):
4551 - 4563.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Krick, O. Platoshyn, M. Sweeney, H. Kim, and J. X.-J. Yuan
Activation of K+ channels induces apoptosis in vascular smooth muscle cells
Am J Physiol Cell Physiol,
April 1, 2001;
280(4):
C970 - C979.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Gao, Y. Pu, K. Q. Luo, and D. C. Chang
Temporal relationship between cytochrome c release and mitochondrial swelling during UV-induced apoptosis in living HeLa cells
J. Cell Sci.,
January 8, 2001;
114(15):
2855 - 2862.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. I. Kruman, C. Culmsee, S. L. Chan, Y. Kruman, Z. Guo, L. Penix, and M. P. Mattson
Homocysteine Elicits a DNA Damage Response in Neurons That Promotes Apoptosis and Hypersensitivity to Excitotoxicity
J. Neurosci.,
September 15, 2000;
20(18):
6920 - 6926.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. M. Luetjens, N. T. Bui, B. Sengpiel, G. Munstermann, M. Poppe, A. J. Krohn, E. Bauerbach, J. Krieglstein, and J. H. M. Prehn
Delayed Mitochondrial Dysfunction in Excitotoxic Neuron Death: Cytochrome c Release and a Secondary Increase in Superoxide Production
J. Neurosci.,
August 1, 2000;
20(15):
5715 - 5723.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Deshmukh, K. Kuida, and E. M. Johnson , Jr.
Caspase Inhibition Extends the Commitment to Neuronal Death Beyond Cytochrome c Release to the Point of Mitochondrial Depolarization
J. Cell Biol.,
July 11, 2000;
150(1):
131 - 144.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Ackerley, A. J. Grierson, J. Brownlees, P. Thornhill, B. H. Anderton, P. N. Leigh, C. E. Shaw, and C. C.J. Miller
Glutamate Slows Axonal Transport of Neurofilaments in Transfected Neurons
J. Cell Biol.,
July 11, 2000;
150(1):
165 - 176.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Lankiewicz, C. Marc Luetjens, N. Truc Bui, A. J. Krohn, M. Poppe, G. M. Cole, T. C. Saido, and J. H. M. Prehn
Activation of Calpain I Converts Excitotoxic Neuron Death into a Caspase-independent Cell Death
J. Biol. Chem.,
May 26, 2000;
275(22):
17064 - 17071.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. R. Khaled, D. A. Reynolds, H. A. Young, C. B. Thompson, K. Muegge, and S. K. Durum
Interleukin-3 Withdrawal Induces an Early Increase in Mitochondrial Membrane Potential Unrelated to the Bcl-2 Family. ROLES OF INTRACELLULAR pH, ADP TRANSPORT, AND F0F1-ATPase
J. Biol. Chem.,
February 23, 2001;
276(9):
6453 - 6462.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. L. Budd, L. Tenneti, T. Lishnak, and S. A. Lipton
Mitochondrial and extramitochondrial apoptotic signaling pathways in cerebrocortical neurons
PNAS,
May 23, 2000;
97(11):
6161 - 6166.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
Compound via MeSH
