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Volume 16, Number 15,
Issue of August 1, 1996
pp. 4696-4706
Copyright ©1996 Society for Neuroscience
Potassium Deprivation-Induced Apoptosis of Cerebellar Granule
Neurons: A Sequential Requirement for New mRNA and Protein
Synthesis, ICE-Like Protease Activity, and Reactive Oxygen
Species
Jörg B. Schulz,
Michael Weller, and
Thomas Klockgether
Department of Neurology, University of Tübingen, D-72076
Tübingen, Germany
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Potassium (K+) deprivation-induced apoptosis
of cerebellar granule neurons requires new mRNA and protein synthesis.
Using a fluorogenic substrate for interleukin-1 converting enzyme
(ICE), we show that K+ deprivation of cerebellar
granule neurons induces cycloheximide-sensitive ICE-like protease
activity. A peptide inhibitor of ICE-like protease activity,
Ac-YVAD-chloromethylketone (Ac-YVAD-CMK), prevents
K+ deprivation-induced apoptosis. Further,
reactive oxygen species (ROS) are essential mediators of
K+ deprivation-induced apoptosis of cerebellar
granule neurons because neuronal death is also blocked by superoxide
dismutase, N-acetyl-L-cysteine, and
free radical spin traps. Using fluorescent assays, we show that ROS
production after K+ deprivation is blocked by
actinomycin D, cycloheximide, and Ac-YVAD-CMK, suggesting that ROS act
downstream of gene transcription, mRNA translation, and ICE activation.
Taken together, we show that new mRNA and protein synthesis, activation
of ICE-like proteases, and ROS production are sequential events in
K+ deprivation-induced apoptosis of cerebellar
granule neurons.
Key words:
apoptosis;
reactive oxygen species;
interleukin-1
converting enzyme;
ICE inhibition;
cerebellar granule neurons;
spin
traps;
superoxide dismutase
INTRODUCTION
Programmed cell death refers to a spatially and
temporally reproducible loss of cells that occurs during the normal
development of the CNS. Neuronal programmed cell death is thought to
serve the removal of neuronal precursors that fail to establish
appropriate synaptic connections (Oppenheim, 1991 ; Johnson and
Deckwerth, 1993). Morphologically, apoptosis underlies some, but not
all, forms of developmental programmed cell death in the mammalian
brain (Wood et al., 1993). Inappropriate apoptosis has been suggested
to be involved in neuronal loss in various human neurodegenerative
diseases, such as Alzheimer's disease (Loo et al., 1993),
Huntington's disease (Portera-Cailliau et al., 1995), amyotrophic
lateral sclerosis (Rabizadeh et al., 1995), and spinal muscular atrophy
(Roy et al., 1995).
In vitro, mature cerebellar granule neurons deprived of
depolarizing levels (25 mM) of extracellular
potassium (K+) undergo apoptosis characterized by
chromatin condensation, pyknosis, and nucleosomal size DNA
fragmentation (D'Mello et al., 1993; Yan et al., 1994; Galli et al.,
1995). Apoptosis of cerebellar granule neurons after
K+ deprivation is blocked by inhibitors of
macromolecular synthesis, forskolin, and insulin-like growth
factor.
The intracellular events that result in apoptosis are often genetically
controlled by proapoptotic and antiapoptotic genes. The CED3 protein,
which is required for cell death during the development of
Caenorhabditis elegans, shows structural (Walker et al.,
1994; Wilson et al., 1994) and functional (Miura et al., 1993; Yuan et
al., 1993) homology to mammalian interleukin-1 converting enzyme
(ICE). ICE is the first member of a family of cysteine proteases with
the distinguishing feature of a near-absolute specificity for aspartate
in the S1 subsite (Thornberry et al., 1992).
Subsequently, further proteases of the ICE familiy were identified as
ICE-CED-3 homolog 1 (ICH-1/NEDD2), ICH-2, or CPP32/YAMA (Miura et al.,
1993; Kumar et al., 1994; Wang et al., 1994; Nicholson et al., 1995;
Tewari et al., 1995).
The mammalian homolog of the C. elegans ced-9 gene, which is
a potent suppressor of cell death, is the bcl-2 gene. It has
been suggested that bcl-2 regulates an antioxidant pathway
to prevent apoptosis in lymphocytes (Hockenbery et al., 1993; Kane et
al., 1993). Events that lead to oxidative stress, including exposure to
-amyloid (Loo et al., 1993; Behl et al., 1994) and transient
ischemia (Héron et al., 1993; Rosenbaum et al., 1994; Li et al.,
1995), may trigger neuronal apoptosis.
Reactive oxygen species (ROS) have been implicated as mediators
of excitotoxic (Coyle and Puttfarcken, 1993; Schulz et al., 1995a,b)
and apoptotic (Hockenbery et al., 1993; Kane et al., 1993; Greenlund et
al., 1995; Rabizadeh et al., 1995) neuronal death. Overexpression of
glutathione peroxidase inhibits apoptosis (Hockenbery et al., 1993),
and the overexpression of copper-zinc superoxide dismutase (SOD) has
been shown to inhibit apoptosis both in neural cell lines (Rabizadeh et
al., 1995) and in primary neurons in culture (Greenlund et al., 1995).
Free radicals may serve either as effectors of cell death, resulting in
oxidative damage of DNA, lipids, and proteins, or as signaling
molecules via redox-sensitive cellular factors such as c-jun
or NF B (Bredesen, 1995)
Here we report that new mRNA and protein synthesis, induction of
ICE-like activity, and formation of ROS are sequential steps in
K+ deprivation-induced apoptosis of cerebellar
granule neurons.
MATERIALS AND METHODS
Materials. Actinomycin D, cycloheximide,
N-tert-butyl- -phenylnitrone (PBN), SOD,
N-acetyl-L-cysteine, vitamin E,
glutathione, catalase,
cytosine--D-arabinofuranoside (Ara-C),
phenylmethylsulfonyl fluoride (PMSF),
N-tosyl-L-lysyl chloromethylketone
(TLCK), N-tosyl-L-phenylalanyl
chloromethylketone (TPCK), E-64, aprotinin, pepstatin,
poly-(L-lysine), and fluorescein diacetate were
obtained from Sigma (St. Louis, MO). Biotinylated anti-rabbit IgG,
control rabbit IgG, peroxidase, and alkaline phosphatase-conjugated
anti-rabbit IgG were from Dakopatts (Glostrup, Denmark). RNase,
avidin-alkaline phosphatase, fluorescein isothiocyanate-streptavidin,
nitroblue tetrazolium chloride, and 5-bromo-4-chloro-3-indolyl
phosphate were purchased from Boehringer Mannheim (Mannheim, Germany).
Ac-YVAD-chloromethylketone (Ac-YVAD-CMK) and DABCYL-YVADAPV-EDANS were
purchased from Bachem (Heidelberg, Germany).
2 ,7-Dichlorodihydrofluorescein diacetate (DCF-H2) and
dihydrorhodamine 123 were obtained from Molecular Probes (Groningen,
Netherlands). ICE p10 (M20) rabbit polyclonal antibody raised against
the p10 subunit of murine ICE was purchased from Santa Cruz
Biotechnology (Heidelberg, Germany). p53-antibody (AB-1) was
obtained from Oncogene Science (Cam- bridge, MA) .
Neuronal cultures. Cerebellar granule neurons were prepared
from 8-d-old Sprague-Dawley rat pups (Interfauna, Tuttlingen, Germany)
as described previously (Novelli et al., 1988; Marini and Paul, 1992;
Weller et al., 1992, 1994c). Cells were dissociated from freshly
dissected cerebella by mechanical disruption in the presence of trypsin
and DNase and then plated in
poly-L-lysine-precoated 35 or 100 mm culture
plates (Nunc, Wiesbaden, Germany) or 24-well plates. Cells were seeded
at a density of 2.1 × 105
cells/cm2 in basal modified Eagle's (BME) medium
supplemented with 10% fetal calf serum, 2 mM
glutamine, and 20 µg/ml gentamycin. Cells maintained in depolarizing
conditions were supplemented with 20 mM KCl to
achieve a final concentration of 25 mM
K+. Ara-C (10 µM) was
added to the culture medium after 24 hr to arrest the growth of
non-neuronal cells. Cultures were fed 5 mM
D-glucose on day in vitro (DIV) 7. Cultures generated by this method have been characterized and shown to
contain >95% granule neurons (Nicoletti et al., 1986; M. Weller,
unpublished data).
Treatment of cultures. All experiments were performed using
cerebellar granule neurons at DIV 8. Neuronal viability was
determined 24 hr later. Culture medium was replaced with serum-free BME
medium containing 5 mM KCl and supplemented with
glutamine and gentamycin as indicated above. Control cells were treated
identically but maintained in serum-free BME medium supplemented with
25 mM KCl (final concentration). All drug
solutions were sterilized by filtration and added in a volume of 2-20
µl.
Morphometric analysis of cell viability. Neurons plated in
35 mm dishes were used for assessment of viability. Neuronal viability
was assessed by the capability of cells to diesterify and retain
fluorescein diacetate in their cytoplasm (Marini and Paul, 1992; Weller
et al., 1992). Cells were washed with Locke's buffer (in
mM): 154 NaCl, 5.6 KCl, 2.3 CaCl2, 1 MgCl2, 3.6 NaHCO3, 5 HEPES, and 20 glucose, incubated for 3 min at 37°C with 5 µg/ml fluorescein diacetate dissolved in
Locke's buffer, washed with Locke's buffer again, and then examined
under ultraviolet light microscopy. Three randomly chosen fields from
each dish were digitized by a blinded observer using a CCD camera
connected to an image processor (MCID-IV, Imaging Research, St.
Catharine's, Ontario, Canada). Images were averaged, filtered, and the
total number of cells was estimated automatically by MCID-IV computer
software. Parameters for the detection of granule neurons were the
shape of cells (round) and the average diameter of the granule neurons
(7-12 µm). We confirmed the accuracy of computerized granule-neuron
counting after switch to either high K+ or low
K+ serum-free medium by comparing the counts of a
blinded observer with the counts of the computer in several
experiments. In high K+ controls, 1500-1800
cerebellar neurons were counted per field. Viability is expressed as
percentage of cerebellar granule neurons retaining fluorescein compared
with high K+ control cultures treated with the
same drug.
Analysis of production of ROS. Cerebellar granule neurons
were seeded in 24-well plates at a density of 2.1 × 105 cells/cm2 as described
above. After the switch to medium containing low concentrations of KCl
(5 mM) at DIV 8, the cultures were incubated for
30 min with 2 µg/ml dihydrorhodamine 123 or 1 µg/ml
DCF-H2 at the time points indicated. Cells were washed with
Locke's buffer and read on a CytoFluor 2350 plate reader (Millipore,
Bedford, MA) at 485 nm excitation and 530 nm emission. Neurons that
were not switched did not show any fluorescence and were used for
background readings.
Fluorometric quantification of DNA fragmentation. For
quantitative DNA fluorometry (Weller et al., 1994a,b), detached
cerebellar granule neurons were harvested and pooled with the attached
cells. All cells were lysed in 10 mM Tris-HCl, pH
7.5, 10 mM EDTA, and 0.2% Triton X-100 for 10 min on ice. Fragmented DNA was separated from nucleus-attached DNA by
high-speed centrifugation. After disruption of the pellets by brief
sonication and RNase A digestion, fragmented and pelleted DNA was
measured by ethidium bromide (0.5 µg/ml) fluorometry using 530 nm
excitation and 620 nm emission wavelengths (Cyto- Fluor 2350). The
linear range was between 0.05 and 3 µg/ml DNA. Percent fragmentation
was calculated by dividing fragmented DNA by the total sum of
fragmented and pelleted DNA.
Detection of ICE and p53 expression. Expression of ICE was
detected by Western blot (Weller et al., 1994b, 1995). Cerebellar
granule neurons were seeded on 100 mm dishes. Soluble protein was
harvested from cells lysed for 15 min on ice in 50 mM Tris-HCl, pH 8, containing 120 mM NaCl, 0.5% NP-40, 2 µg/ml aprotinin, 100 µg/ml PMSF, 10 µg/ml leupeptin, 50 mM sodium
fluoride, and 200 µM sodium vanadate followed
by high-speed centrifugation at 4°C. Twenty micrograms protein per
lane were separated by 15% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and electroblotted to nitrocellulose. Immunodetection
involved blocking for 1 hr in 10 mM Tris-HCl, pH
7.5, containing 150 mM NaCl, 0.1% Tween 20, 5%
skim milk, and 2% BSA; incubation with antibodies to ICE p10 (2 µg/ml) or p53 (2 µg/ml) overnight at 4°C; incubation with
biotinylated anti-rabbit IgG (1:20,000 in PBS/0.1% Tween 20),
streptavidin-alkaline phosphatase (1:1,000), and nitroblue tetrazolium
chloride (0.41 mM), and
5-bromo-4-chloro-3-indolyl phosphate (0.38 mM) in
200 mM Tris-HCl, pH 9.5, containing 10 mM MgCl2 as substrate.
ICE activity. Cerebellar granule neurons were seeded in
24-well plates at a density of 2.1 × 105
cells/cm2 as described above. After the switch to
medium containing low (5 mM) or high (25 mM) concentrations of KCl at DIV 8, cerebellar
granule neurons were washed with Locke's buffer and made permeable by
0.03% digitonin for analysis of ICE-like protease activity at time
points indicated. After 10 min, the fluorogenic ICE substrate
DABCYL-YVADAPV-EDANS (20 µM) was added.
Fluorescence was determined in 10 min intervals for 1 hr using 360 nm
excitation and 480 nm emission wavelengths (CytoFluor 2350) in the
24-well plates. The fluorometric intensity peaked at 20 min. This time
point was used for statistical analysis.
Statistics. Data are expressed as mean ± SEM.
Statistical significance was assessed by two-tailed Student's
t test (comparison of 2 groups) or one-way ANOVA followed by
Scheffe's post hoc test (comparison of more than 2 groups). All
experiments reported here represent at least three independent
replications performed in triplicate.
RESULTS
K+ deprivation-induced apoptosis of cerebellar
granule neurons
Apoptosis of differentiated cerebellar granule neurons can be
induced by lowering the extracellular K+
concentration from 25 to 5 mM DIV 8 (D'Mello et
al., 1993; Yan et al., 1994; Galli et al., 1995). Switch to low
K+ concentrations decreases viability of
cerebellar granule neurons by >50% when measured after 24 hr. As
previously reported (Yan et al., 1994), exposure to the transcriptional
inhibitor actinomycin D and the translational inhibitor cycloheximide
at the time of K+ deprivation protected against
low K+-induced neuronal death as assessed by
staining with fluorescein diacetate at 24 hr (Fig. 1,
Table 1). Quantitative DNA fluorometry showed that at
this time point 28 ± 3% of the total DNA was fragmented in
neurons maintained in low K+ versus 10 ± 2% maintained in high K+ medium. Treatment with
actinomycin D and cycloheximide prevented DNA fragmentation induced by
K+ withdrawal (Table 1). Thus, new mRNA and
protein synthesis appear to be required for K+
deprivation-induced apoptosis of cerebellar granule neurons.
Fig. 1.
Cycloheximide and PBN prevent low
K+ deprivation-induced apoptosis. Neurons at DIV
8 were switched from high K+ (25 mM) to low K+ (5 mM) serum-free culture medium and stained for
viable cells with fluorescein diacetate 24 hr later. Stained cells were
visualized under ultraviolet illumination and digitized using a CCD
camera. Culture A was maintained in 25 mM, cultures B-D were kept at 5 mM K+. Low
K+ neurons were either untreated (B)
or treated with 10 µg/ml cycloheximide (C) or 100 µM PBN (D). Scale bar, 50 µm.
[View Larger Version of this Image (171K GIF file)]
Table 1.
Effects of actinomycin D and cycloheximide on
K+ deprivation-induced apoptosis of cerebellar granule
neurons
| Treatment |
Medium |
Viability [%] |
DNA fragmentation [%]
|
|
| No |
high K+ |
100
± 3 |
10 ± 2 |
| No |
low K+ |
42 ± 4 |
28
± 3 |
| Actinomycin D (1 µg/ml) |
high K+ |
100
± 6 |
11 ± 2 |
| Actinomycin D (1 µg/ml) |
low
K+ |
89 ± 5* |
8 ± 1* |
| Cycloheximide (10 µg/ml) |
high K+ |
100 ± 5 |
8 ± 2
|
| Cycloheximide (10 µg/ml) |
low K+ |
92
± 5* |
9 ± 1* |
|
|
Cultures were switched to high (25 mM) or low (5 mM) K+ serum-free medium, and actinomycin D or
cycloheximide was added at concentrations indicated. Viability was
determined by fluorescein diacetate staining at 24 hr. DNA
fragmentation was determined by quantitative DNA fluorometry 24 hr
after switch to low or high K+ medium. *p < 0.001 compared with untreated cerebellar granule neurons switched to
low K+ concentrations.
|
|
p53 and ICE protein expression in low K+-induced
apoptosis of cerebellar granule neurons
We studied the role of two putative mediators of
apoptotic cell death by Western blot. The tumor suppressor gene product
p53 mediates apoptosis after DNA damage in certain cell types (Clarke
et al., 1993; Lowe et al., 1993a,b). Although DNA breaks and DNA
fragmentation are characteristic features of K+
deprivation-induced apoptosis of cerebellar granule neurons, we did not
find any expression of p53 protein by Western blot up to 24 hr after
K+ deprivation (Fig.
2A). T98G human malignant glioma cells, which
express abundant mutant p53, were used as a positive control (Ullrich
et al., 1992).
Table 2.
Effects of protease inhibitors on K+
deprivation-induced apoptosis of cerebellar granule neurons
| Inhibitor |
Specificity |
Medium |
Viability
[%] normalized to high K+ (25 mM)
|
| No
treatment |
[1 µM] |
[10 µM] |
[100
µM] |
|
| TPCK |
Chymotrypsin |
high
K+ |
100 ± 5 |
93 ± 5 |
20
± 8 |
0 |
|
|
low K+ |
59
± 6 |
57 ± 2 |
15 ± 9 |
0 |
| TLCK |
Trypsin |
high
K+ |
100 ± 5 |
95 ± 7 |
83 ± 4 |
0
|
|
|
low K+ |
59 ± 6 |
52 ± 4 |
48
± 5 |
0 |
| PMSF |
Serine protease |
high K+ |
100
± 8 |
n.t. |
101 ± 7 |
70 ± 1 |
|
|
low
K+ |
55 ± 7 |
n.t. |
41 ± 6 |
57 ± 4
|
| E-64 |
Cysteine protease |
high K+ |
100
± 5 |
n.t. |
104 ± 5 |
92 ± 7 |
|
|
low
K+ |
59 ± 6 |
n.t. |
61 ± 7 |
57 ± 5
|
| Leupeptin |
Cysteine proteases |
high K+ |
100
± 5 |
n.t. |
99 ± 3 |
107 ± 7 |
|
|
low
K+ |
47 ± 5 |
n.t. |
60 ± 4 |
56 ± 5
|
| Pepstatin |
Aspartic proteases |
high K+ |
100
± 5 |
n.t. |
99 ± 4 |
66 ± 7 |
|
|
low
K+ |
59 ± 6 |
n.t. |
52 ± 6 |
33
± 2 |
|
|
Cultures were preincubated with the protease inhibitors for 1 hr.
After switch to high (25 mM) or low (5 mM)
K+ serum-free medium protease, inhibitors were readded at
concentrations indicated. Viability was determined by fluorescein
diacetate staining. None of the treatments was protective. TPCK and
TLCK were toxic to cerebellar granule cells in low K+ and
high K+ medium at concentrations of 10 and 100 µM, respectively. n.t., Not tested.
|
|
Fig. 2.
p53 and ICE protein expression of cerebellar
granule neurons during K+ deprivation-induced
apoptosis. Protein expression was determined at the time points
indicated after switch to serum-free medium with high (25 mM) or low (5 mM)
concentrations of K+. A, p53 protein
expression was not detected in cerebellar granule neurons after switch
to low or high K+ medium. T98G human malignant
glioma cells served as a positive control. B, The 45 kDa ICE
precursor protein, but not the cleaved 10 kDa protein, was detected in
cerebellar granule neurons.
[View Larger Version of this Image (35K GIF file)]
The cysteine protease ICE is the mammalian homolog of CED-3, a C. elegans gene product required for cell death (Yuan et al., 1993).
Although we detected the 45 kDa ICE precursor (proICE) protein by
immunoblot analysis with an antibody directed against the p10 subunit
of murine ICE, we did not observe a band corresponding to the cleaved
and active 10 kDa subunit or a decrease in expression of the 45 kDa
proICE-protein, arguing against an activation of ICE (Fig.
2B).
Inhibition of ICE-like proteases protects against apoptosis of
cerebellar granule neurons
To screen for other ICE-like proteases that might be involved in
low K+-induced apoptosis of cerebellar granule
neurons, we used Ac-YVAD-CMK, an irreversible and specific peptide
inhibitor of ICE and ICE-like proteases (Thornberry et al., 1992;
Lazebnik et al., 1994; Enari et al., 1995). Figure 3
shows that Ac-YVAD-CMK attenuated low K+-induced
apoptosis of cerebellar granule neurons in a concentration-dependent
manner when added at the time of K+ withdrawal.
Additional preincubation with Ac-YVAD-CMK for 1 hr resulted in almost
complete survival of cerebellar granule neurons as assessed 24 hr after
K+ deprivation (Fig. 3A). The peptidic
nature of the ICE inhibitor limits cell penetration, which may explain
the requirement of micromolar concentrations and preincubation with
this agent for complete inhibition of apoptosis. Treatment with
Ac-YVAD-CMK had no effect on granule neurons maintained at high
K+ concentrations, indicating that the ICE
inhibitor is not toxic at concentrations tested. The cysteine protease
inhibitors E-64 and leupeptin, the aspartic protease inhibitor
pepstatin, the serine protease inhibitor PMSF, the chymotrypsin
inhibitor TPCK, and the trypsin inhibitor TLCK had no effect on
K+ deprivation-induced apoptosis at
concentrations tolerated by the granule neurons (Table 2).
Fig. 3.
The role of ICE-like proteases in
K+ deprivation-induced apoptosis. A,
Inhibition of ICE-like protease activity abrogates
K+ deprivation-induced apoptosis of cerebellar
granule neurons. Apoptosis was induced by K+
deprivation. Neurons maintained in high K+ medium
were used to control for possible toxic effects of the ICE inhibitor.
Neuronal viability was assessed by fluorescein diacetate staining and
interactive counting by an image analysis system. The ICE inhibitor
tetrapeptide Ac-YVAD-CMK was added at the time point of medium switch.
Additional preincubation with Ac-YVAD-CMK for 1 hr before switch showed
the best protection (100 µM × 2).
***p < 0.001, **p < 0.01, *p < 0.05 compared with untreated granule neurons
maintained at low K+. B, ICE-like
protease activity is induced after K+
deprivation. ICE-like protease activity was measured with the
fluorogenic ICE substrate DABCYL-YVADAPV-EDANS in the absence or
presence of Ac-YVAD-CMK in low or high K+ medium
at defined time points after medium switch.
[View Larger Version of this Image (24K GIF file)]
To confirm that K+ deprivation induces ICE-like
proteases, ICE-like proteolytic activity was measured using the
fluorogenic ICE substrate
4-(4-dimethylaminophenylazo)benzoyl-YVADAPVU-5-[(2-aminoethyl)amino]-naphthalene-1-sulfonic
acid (DABCYL-YVADAPV-EDANS), which contains the cleavage
site of the ICE-like enzymes (Pennington and Thornberry, 1994).
Measurements at 1, 2, and 3 hr after switch to low
K+ confirmed an increase in ICE-like protease
activity that was blocked by Ac-YVAD-CMK (Fig. 3B).
Antioxidants and free radical spin traps block K+
deprivation-induced apoptosis of cerebellar granule neurons
ROS have been implicated as important effector molecules in
apoptotic cell death (Hockenbery et al., 1993; Kane et al., 1993;
Zamzami et al., 1995). Therefore, we studied the effects of the
antioxidants SOD, N-acetyl-L-cysteine,
catalase, glutathione, vitamin E, and PBN on apoptosis of cerebellar
granule neurons induced by K+ deprivation at DIV
8. PBN is a free radical spin trap that reacts with free radicals to
form stable adducts and can therefore serve as a free radical scavenger
(Knecht and Mason, 1993; Schulz et al., 1995a). Exposure to PBN, SOD,
and N-acetyl-L-cysteine concentration
dependently inhibited cerebellar granule neuron death at 24 hr (Fig.
4). Treatment with glutathione (100 µM: 88 ± 5% vs 51 ± 4% viability
normalized to 25 mM K+;
p < 0.01), vitamin E (1 mg/ml: 78 ± 6% vs
51 ± 4%; p < 0.05), and catalase (1000 U/ml:
72 ± 5% vs 51 ± 4%; p < 0.05) was also
effective.
Fig. 4.
K+ deprivation-induced
apoptosis requires ROS formation. Effects of treatment with
N-tert-butyl--phenylnitrone (PBN)
(A), superoxide dismutase (SOD) (B),
and N-acetyl-L-cysteine (C).
Neuronal viability was assessed by fluorescein diacetate staining and
interactive counting by an image analysis system at 24 hr after
switching to low K+. ***p < 0.001, **p < 0.01, *p < 0.05 compared
with untreated cells switched to low K+.
D shows the time course of ROS production as detected by
dihydrorhodamine 123 fluorescence. Because the switch to serum-free
high K+ medium led to a minor increase in the
fluorescence of both dyes compared with nonswitched controls, the
difference in fluorescence between high K+- and
low K+-treated cultures at every time point is
given.
[View Larger Version of this Image (39K GIF file)]
To confirm the generation of ROS directly and to follow the time course
of ROS production, we used the oxidation-sensitive indicators
dihydrorhodamine 123 and DCF-H2. These lipophilic
nonfluorescent indicators are easily deacetylated to their active forms
after penetrating cells. They are oxidized by ROS to the fluorescent
dyes rhodamine 123 and DCF, respectively. Because the fluorescent
derivative rhodamine 123 is positively charged, it moves to the
inside-negative mitochondrial environment (Johnson et al., 1980).
Staining with dihydrorhodamine 123 showed a gradual increase in
fluorescence over time with a maximum at 4 hr after
K+ deprivation and a slow decline thereafter
(Fig. 4D). A similar time course of ROS production with a
maximum at 4 hr was obtained by staining with DCF-H2 with
the exception of an additional minor peak of DCF fluorescence at 1 hr.
Four hours after switch to low K+ concentrations,
we used confocal laser microscopy to confirm that ROS were generated
intracellularly. Culture dishes were loaded with 4 µg/ml
dihydrorhodamine 123 or 2 µg/ml DCF-H2 for 30 min.
After this incubation period, cellular fluorescence was imaged using a
laser scanning confocal microscope at 485 nm excitation and 530 nm
emission. For both dyes, fluorescence was strictly localized within
cerebellar granule neurons (data not shown).
DNA fragmentation and survival
Fragmentation of nuclear DNA is a typical feature of apoptosis. A
17% increase of DNA fragmentation was observed at 24 hr after switch
to low K+ medium compared with switch to high
K+ medium (Fig. 5A).
This increase was blocked by treatment with actinomycin D and
cycloheximide, but not with PBN or Ac-YVAD-CMK. Yet, these drugs
greatly enhanced neuronal viability as assessed by staining with
fluorescein diacetate at the same time point. To confirm that PBN- and
Ac-YVAD-CMK-mediated rescue of staining with fluorescein diacetate at
24 hr after K+ deprivation truly reflected
survival compared with untreated neurons, and that DNA fragmentation at
24 hr did not inevitably predict neuronal death, cerebellar granule
neurons were switched back to their initial conditioned medium 24 hr
after K+ deprivation and cultured for another 48 hr. According to staining with fluorescein diacetate, reexposure to the
original medium arrested the process of neuronal apoptosis induced by
K+ deprivation in that neuronal counts exceeded
35% in cultures switched back to high K+ medium,
whereas <10% of the neurons survived 72 hr culture in low
K+ medium (Fig. 5B). Further, staining
with fluorescein diacetate at 72 hr confirmed near-complete rescue from
K+ deprivation-induced apoptosis in neurons
exposed to either cycloheximide, ICE-inhibitor, or PBN during
K+ deprivation from 0 to 24 hr. At 72 hr, DNA
fragmentation in neurons treated with PBN and Ac-YVAD-CMK was reduced
compared with measurements at the end of K+
deprivation (24 hr). In fact, DNA fragmentation of PBN- or
Ac-YVAD-CMK-treated neurons at 72 hr was not different from that of
cycloheximide-treated neurons (Fig. 5A), suggesting that DNA
repair takes place during high K+-mediated rescue
of the neurons between 24 and 72 hr. Because there was no decrease in
total DNA harvests between 24 and 72 hr, a selective loss of fragmented
DNA into the culture medium is unlikely to account for the decrease of
DNA fragmentation.
Fig. 5.
DNA fragmentation and survival after
K+ deprivation. Cell viability and specific DNA
fragmentation were measured 24 (A) and 72 (B) hr
after switch to low K+ medium. Specific DNA
fragmentation was calculated by subtracting baseline DNA fragmentation
before calculating ratios of fragmented versus pelleted DNA in
experimental samples. In these experiments, baseline DNA fragmentation
never exceeded 9% in cells switched to high K+
concentrations. A, Treatment with cycloheximide
(CHX), Ac-YVAD-CMK, and PBN
greatly enhanced neuronal viability at 24 hr after
K+ withdrawal as assessed by staining with
fluorescein diacetate. However, only treatment with cycloheximide
prevented DNA fragmentation (***p < 0.001).
B, When cerebellar granule neurons were switched back to
their initial conditioned medium 24 hr after K+
deprivation and cultured for another 48 hr, significantly more neurons
remained viable compared with neurons continuously maintained in low
K+ medium for 72 hr (p < 0.01). Furthermore, treatment with cycloheximide, ICE inhibitor, and
PBN was protective. After switch back to preconditioned high
K+ medium, DNA fragmentation was markedly reduced
in treated and untreated cells compared with neurons at the end of
K+ deprivation (24 hr) and with neurons
maintained in low K+ for 72 hr
(**p < 0.01).
[View Larger Version of this Image (25K GIF file)]
Sequential mechanisms of apoptosis in cerebellar
granule neurons
To characterize the sequential mechanisms during
K+ deprivation-induced apoptosis of cerebellar
granule neurons, we determined the point of commitment to death as the
latest time at which neurons can be rescued after switch to low
K+ (5 mM) by addition of a
given survival agent (Fig. 6). Neurons were shifted to
low K+ medium and, at different time points,
actinomycin D, cycloheximide, or PBN was added to the culture medium.
At 24 hr, neuronal survival was evaluated by fluorescein diacetate
staining. Neurons were rescued by addition of PBN up to 3 hr after the
initial switch to low K+. The therapeutic window
for the transcriptional and translational inhibitors actinomycin D and
cycloheximide, respectively, was shorter. To obtain a significant
protection, actinomycin D had to be added within 40 min and
cycloheximide within 60 min after K+ deprivation.
These results suggest that, within 1 hr, most granule neurons have
transcribed and translated the message for one or more putative
``suicide proteins'' and that ROS are produced downstream of this
protein synthesis. Because the peptide ICE inhibitor required prolonged
exposure for cell protection, no such experiment for the kinetics of
ICE inhibitors could be performed.
Fig. 6.
Effects of delayed treatment to prevent apoptosis
triggered by K+ deprivation of cerebellar
granule neurons. Rescue treatments included actinomycin D (1 µg/ml),
cycloheximide (10 µg/ml), and PBN (100 µM) at
the time points indicated. Neuronal viability was evaluated by staining
with fluorescein diacetate and automated counting by image analyzing
software. A significant protection (p < 0.01, ANOVA) was provided by actinomycin D up to 40 min, cycloheximide up to
1 hr, and PBN up to 3 hr.
[View Larger Version of this Image (19K GIF file)]
To further elucidate the sequential mechanisms of apoptosis in
cerebellar granule neurons, we studied the effects of cycloheximide,
Ac-YVAD-CMK, and antioxidants on ICE-like protease activity as measured
with the fluorogenic ICE substrate DABCYL-YVADAPV-EDANS. As expected,
Ac-YVAD-CMK completely blocked ICE-like activity (Fig.
7A). In addition, neurons treated with
cycloheximide at the time of switching did not exhibit ICE-like
activity, suggesting that the putative killer proteins are an upstream
positive regulator of ICE-like protease activity. In contrast, PBN,
SOD, and N-acetyl-L-cysteine did not
affect ICE-like protease activity. In another experiment, we studied
the effects of cycloheximide, Ac-YVAD-CMK, and PBN on ROS production
with the oxidation-sensitive indicators DCF-H2 and
dihydrorhodamine 123 at 4 hr after K+ deprivation
(Fig. 7B,C). As expected, PBN prevented the increase of DCF
and rhodamine 123 fluorescence. However, cycloheximide and Ac-YVAD-CMK
had a similar effect, suggesting that both agents interfere with the
apoptotic cascade upstream of ROS formation.
Fig. 7.
Interaction of RNA translation, induction of
ICE-like protease activity, and formation of ROS. A,
Cycloheximide (CHX) and Ac-YVAD-CMK, but
not PBN, SOD, or
N-acetyl-L-cysteine (N-AC)
prevent the induction of ICE-like activity as measured with the
ICE-substrate DABCYL-YVADAPV-EDANS at 3 hr after
K+ deprivation. Cycloheximide
(CHX), Ac-YVAD-CMK, and PBN
attenuate the production of ROS as detected by 2,7-DCF (B)
and rhodamine 123 (C) fluorescence at 4 hr after switch to
low K+ medium. **p < 0.01, ***p < 0.001 compared with untreated granule neurons
switched to low K+ medium.
[View Larger Version of this Image (19K GIF file)]
DISCUSSION
The regulation of programmed cell death in the developing nervous
system involves target-derived survival factors, afferent synaptic
activity, and hormone- and cytokine-dependent signaling. Cerebellar
granule neurons undergo extensive cell death characterized by nuclear
DNA fragmentation between postnatal days 5 and 9 (P5 and P9) in
vivo (Wood et al., 1993). Mossy fiber input to the granule cell
layer begins at P5, and synapse formation occurs by P12 (Burgoyne and
Cambray-Deakin, 1988). Similarly, formation of synaptic contacts
between parallel fibers and Purkinje cell dendrites is prominent
between the second and third postnatal weeks, corresponding to the
maximal rate of synaptogenesis (Burgoyne and Cambray-Deakin, 1988).
Therefore, cell death before synaptogenesis may help to regulate
granule neuron number.
In the absence of exogenously added growth or survival factors other
than those present in fetal calf serum, cerebellar granule neurons can
be differentiated and maintained in vitro for weeks in the
presence of high concentrations of K+. These
neurons undergo highly synchronous apoptosis when deprived of
depolarizing concentrations of K+ (D'Mello et
al., 1993; Yan et al., 1994; Galli et al., 1995). We have previously
suggested that the functional innervation of postmigratory granule
neurons during cerebellar development by mossy fibers and climbing
fibers may prevent further elimination of these neurons by blocking
their programmed death (Yan et al., 1994). Therefore,
K+ deprivation-induced apoptosis of
differentiated cerebellar granule neurons may be a model of neuronal
death after deafferentation. On the other hand, studies that examined
the postnatal histogenesis of the cerebellum in normal mice,
neurologically mutant mice, and chimeras between normal and mutant mice
show that there is a numerical matching of granule neurons with
Purkinje cells. They indicate that a target-related cell death of
granule cells occurs during development of the cerebellum. The
survival-promoting effects of K+ in cortical
neurons are mediated by a specific neurotrophin, brain-derived
neurotrophic factor (Ghosh et al., 1994), suggesting that
K+ deprivation-induced apoptosis of cerebellar
granule neurons may also involve specific unidentified growth
factors.
A requirement for new mRNA and protein synthesis is a typical feature
of neuronal apoptosis (Johnson and Deckwerth, 1993). Apoptosis of
several non-neuronal cells, including lymphoid cells and neoplastic
cells, in contrast, is induced or augmented by inhibitors of mRNA and
protein synthesis (Weller et al., 1994a,b). Here we report that new
mRNA and protein synthesis, ICE-like proteases, and production of ROS
are necessary and sequential events of apoptosis in this paradigm of
neuronal apoptosis. Time-kinetic studies revealed that gene activation
required for apoptosis is an early event, because exposure to
actinomycin D and cycloheximide >60 min after K+
withdrawal failed to enhance neuronal survival when assessed at 24 hr.
This is in contrast to nerve growth factor (NGF) deprivation-induced
apoptosis of sympathetic neurons, 50% of which are rescued when
protein synthesis was blocked within 22 hr of NGF deprivation
(Martin et al., 1992) .
ICE-like proteases have recently been identified as important mediators
of apoptotic cell death, e.g., in Fas/APO-1-mediated apoptosis (Enari
et al., 1995; Kuida et al., 1995; Los et al., 1995) and programmed cell
death of motor neurons (Milligan et al., 1995). The detection of
ICE-like activity by a substrate peptide and the protective effects of
an inhibitor of ICE-like proteases strongly suggest that a member of
the ICE family of proteases is also involved in
K+ deprivation-induced apoptosis of cerebellar
granule neurons. Treatment with other chloromethylketone inhibitors of
proteases (TPCK and TLCK) that lack aspartate in the P1 position and
with other protease inhibitors did not result in enhanced neuronal
survival. We believe that ICE-like protease activity operates
downstream of the putative killer genes because the induction of
enzymatic activity was blocked by cycloheximide.
The fluorogenic ICE substrate DABCYL-YVADAPV/EDANS and the peptide ICE
inhibitor Ac-YVAD-CMK do not differentiate between members of the ICE
family (Lazebnik et al., 1994; Nicholson et al., 1995; Tewari et al.,
1995). To examine whether ICE or other ICE-like proteases mediate
K+ deprivation-induced apoptosis, we studied the
role of ICE by Western blotting. Mammalian ICE is synthesized as an
inactive 45 kDa precursor processed proteolytically to generate active
enzyme that comprises polypeptides of 20 kDa (p20) and 10 kDa (p10)
(Thornberry et al., 1992). We detected the expression of the 45 kDa
precursor protein in cerebellar granule neurons but did not observe a
10 kDa protein or a decrease of the precursor protein up to 8 hr after
K+ deprivation, indicating that the precursor
protein is not cleaved to the active ICE subunits. The p10 antibody we
used was directed against the mouse p10 subunit. The mouse and rat p10
subunits show 95% homology (Keane et al., 1995). The detection of the
45 kDa precursor protein appears to prove that the antibody detects rat
ICE. In contrast, the p10 subunits of other members of the ICE-like
protease family show less homology (Kumar, 1995). Our results suggest
that not ICE itself, but one or more other members of the ICE family
are important regulators of apoptosis in cerebellar granule
neurons.
ROS have been implicated as mediators of excitotoxic (Coyle and
Puttfarcken, 1993; Schulz et al., 1995a,b) and apoptotic (Hockenbery et
al., 1993; Kane et al., 1993; Greenlund et al., 1995; Rabizadeh et al.,
1995) neuronal cell death. One major aim of this study was to examine
the role of ROS in K+ deprivation-induced
apoptosis of cerebellar granule neurons. Using redox-sensitive dyes, we
found that ROS are generated during apoptosis and that inhibitors of
ROS formation prevented neuronal death. Scavengers of superoxide (SOD,
PBN) were equally effective as scavengers of peroxides (glutathione,
catalase), antioxidants (vitamin E), and
N-acetyl-L-cysteine, a thiol
antioxidant and glutathione precursor. These pharmacological data
indicate that superoxide, hydrogen peroxide, and hydroxyl radicals are
produced in K+-deprived cerebellar granule
neurons. Measurements with the redox-sensitive dyes DCF-H2
and dihydrorhodamine 123 indicate that there is an increase in the
formation of ROS that peaks at 4 hr and slowly declines thereafter. The
time course of the production of free radicals correlates well with the
ability of PBN to protect against apoptosis. ROS formation is a
downstream event in granule neuron apoptosis because both cycloheximide
and the ICE inhibitor prevented ROS formation.
The antiapoptotic properties of the bcl-2 oncogene product
have been attributed to the detoxification of ROS (Hockenbery et al.,
1993). Although cerebellar granule neurons maintained in high
K+ medium express bcl-2 mRNA (Montpied
et al., 1993), we have not been able to detect the BCL-2 protein in
cerebellar granule neurons maintained in either high
K+ or low K+ medium (de
Luca et al., 1996). Thus, cerebellar granule neurons may be
specifically vulnerable to ROS-induced toxicity.
ROS formation is a downstream event in the intracellular cascade
leading to apoptosis of cerebellar granule neurons. We show that gene
induction and activation of ICE-like proteases are necessary for the
generation of ROS. The mechanism of ROS-induced cytotoxicity is
unclear. Targets of cell injury by ROS are cellular macromolecules
including DNA. ROS-mediated DNA damage is unlikely to be responsible
for cell death because p53 activity was not induced and because
antioxidants and inhibitors of ICE-like proteases inhibited cell death
but not DNA fragmentation. Apoptosis of neuronal precursors in the
cerebellum of transgenic mice lacking functional p53 is similar to that
in wild-type mice, arguing for a p53-independent apoptotic pathway of
physiological cerebellar granule cell loss during development (Wood and
Youle, 1995). In contrast, p53 expression is required for gamma
irradiation-induced apoptosis of cerebellar granule neuron precursors
in vivo.
In contrast to their recognized role as damaging molecules, ROS have
recently been implicated as important signal transduction molecules.
Sympathetic neurons show an increase in ROS that peaks at 3 hr after
deprivation of NGF. If NGF was added back to the culture medium after
the period of peak ROS generation, apoptosis was completely prevented,
suggesting that ROS production serves as an early signal, rather than a
toxic agent, to mediate apoptosis (Greenlund et al., 1995). Commitment
to cell death is defined as the time after which readdition of trophic
support or other treatment will no longer prevent cell death. Fifty
percent of sympathetic neurons can be rescued when protein synthesis
was blocked 22 hr after NGF deprivation (Martin et al., 1992).
Therefore, it was argued that ROS may signal by modulating
transcription either directly or through known redox-sensitive proteins
or transcription factors. This cascade is different in
K+ deprivation-induced apoptosis of cerebellar
granule neurons, because (1) neurons can only be rescued after
K+ deprivation when inhibitors of transcription
and translation are added during the first hour (whereas free radical
scavengers protect when added up to 3 hr after switch to low
K+) and (2) generation of ROS is a downstream
event of protein synthesis.
Apoptosis of cerebellar granule neurons induced by
K+ deprivation is associated with DNA
fragmentation that is prevented by inhibition of protein synthesis.
However, treatment with the peptide inhibitor of ICE-like proteases,
Ac-YVAD-CMK, and a free radical scavenger did not block DNA
fragmentation at 24 hr after K+ deprivation.
Although 90% of cells were viable at that time point with either
treatment, almost 20% of the total DNA was fragmented. When switched
back to conditioned medium, this fragmentation did not lead to further
cell death, but DNA appeared to be partially repaired. These results
argue against the hypothesis that the degree of DNA fragmentation
observed in cerebellar granule neurons will necessarily lead to
apoptotic death. The finding that apoptosis can be induced in
enucleated cells (cytoblasts) provided evidence that events occurring
independently or upstream of nuclear alterations have a major impact in
the regulation and execution of apoptosis (Jacobson et al., 1994;
Schulze-Osthoff et al., 1994). Our data indicate that DNA fragmentation
is an epiphenomenal event in K+
deprivation-induced apoptosis of cerebellar granule neurons.
Conclusions
Previous studies have shown that K+
deprivation-induced apoptosis of cerebellar granule neurons requires
new mRNA and protein synthesis. Here we present data that provide a
framework for the understanding of the apoptotic cascade in these
neurons. Our results suggest that an ICE-like protease is a critical
mediator of this apoptotic cell death. Although we cannot identify the
specific protease of the ICE family involved in apoptosis of cerebellar
granule neurons, it is unlikely to be ICE itself. Further, ROS are
essential mediators of apoptotic cell death. Because inhibition of
transcription, translation, and of ICE-like activity prevents
production of ROS, ROS formation is a downstream event in the
intracellular cascade leading to apoptotic cell death. The mechanisms
by which ROS induce cell death remain to be elucidated.
FOOTNOTES
Received Feb. 8, 1996; revised April 18, 1996; accepted May 13, 1996.
We thank L. Dumitrescu and I. Müller for excellent technical
assistance and Dr. P.-A. Löschmann for valuable discussions.
Correspondence should be addressed to Dr. Jörg B. Schulz,
Department of Neurology, University of Tübingen,
Hoppe-Seyler-Strasse 3, D-72076 Tübingen,
Germany.
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[Abstract]
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A. G. Yakovlev, S. M. Knoblach, L. Fan, G. B. Fox, R. Goodnight, and A. I. Faden
Activation of CPP32-Like Caspases Contributes to Neuronal Apoptosis and Neurological Dysfunction after Traumatic Brain Injury
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October 1, 1997;
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[Abstract]
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B. A. Eldadah, A. G. Yakovlev, and A. I. Faden
The Role of CED-3-Related Cysteine Proteases in Apoptosis of Cerebellar Granule Cells
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August 15, 1997;
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[Abstract]
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M. Deshmukh and E. M. Johnson Jr.
Programmed Cell Death in Neurons: Focus on the Pathway of Nerve Growth Factor Deprivation-Induced Death of Sympathetic Neurons
Mol. Pharmacol.,
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[Abstract]
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R. C. Armstrong, T. J. Aja, K. D. Hoang, S. Gaur, X. Bai, E. S. Alnemri, G. Litwack, D. S. Karanewsky, L. C. Fritz, and K. J. Tomaselli
Activation of the CED3/ICE-Related Protease CPP32 in Cerebellar Granule Neurons Undergoing Apoptosis But Not Necrosis
J. Neurosci.,
January 15, 1997;
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[Abstract]
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D. Kornitzer, R. Sharf, and T. Kleinberger
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J. Cell Biol.,
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[Abstract]
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M. J. O'Hare, S. T. Hou, E. J. Morris, S. P. Cregan, Q. Xu, R. S. Slack, and D. S. Park
Induction and Modulation of Cerebellar Granule Neuron Death by E2F-1
J. Biol. Chem.,
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[Abstract]
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E. Vereker, V. Campbell, E. Roche, E. McEntee, and M. A. Lynch
Lipopolysaccharide Inhibits Long Term Potentiation in the Rat Dentate Gyrus by Activating Caspase-1
J. Biol. Chem.,
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[Abstract]
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V. See and J.-P. Loeffler
Oxidative Stress Induces Neuronal Death by Recruiting a Protease and Phosphatase-gated Mechanism
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[Abstract]
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A. Hartmann, S. Hunot, P. P. Michel, M.-P. Muriel, S. Vyas, B. A. Faucheux, A. Mouatt-Prigent, H. Turmel, A. Srinivasan, M. Ruberg, et al.
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PNAS,
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[Abstract]
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K.-T. Kim, D.-S. Koh, and B. Hille
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