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The Journal of Neuroscience, February 1, 1998, 18(3):830-840
Multiple Pathways of Neuronal Death Induced by DNA-Damaging
Agents, NGF Deprivation, and Oxidative Stress
David S.
Park1,
Erick
J.
Morris3,
Leonidas
Stefanis1, 2,
Carol M.
Troy1,
Michael L.
Shelanski1,
Herbert M.
Geller3, and
Lloyd A.
Greene1
1 Department of Pathology, Taub Center for Alzheimer's
Disease Research, and 2 Neurology and Center for
Neurobiology and Behavior, Columbia University College of Physicians
and Surgeons, New York, New York 10032, and 3 Department of
Pharmacology, University of Medicine and Dentistry of New Jersey
(UMDNJ)-Robert Wood Johnson Medical School, Piscataway, New Jersey
08854
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ABSTRACT |
Here, we compare the pathways by which DNA-damaging agents,
NGF deprivation, and superoxide dismutase 1 (SOD1) depletion evoke apoptosis of sympathetic neurons. Previous work raised the hypothesis that cell cycle signaling plays a required role in neuronal apoptosis elicited by NGF deprivation and the DNA-damaging agent camptothecin. To
test this hypothesis, we extended our investigation of DNA-damaging agents to cytosine arabinoside (AraC) and UV irradiation. As with NGF
deprivation and camptothecin treatment, the cyclin-dependent kinase
inhibitors flavopiridol and olomoucine protected neurons from apoptosis
induced by AraC and UV treatment. These observations support the model
that camptothecin, AraC, and UV treatment cause DNA damage, which leads
to apoptosis by a mechanism that, as in the case of NGF deprivation,
includes activation of cell cycle components. Flavopiridol and
olomoucine, however, had no effect on death induced by SOD1 depletion,
suggesting that CDKs do not play a role in this paradigm of neuronal
death. To compare further the mechanisms of death evoked by NGF
withdrawal, SOD1 depletion, and DNA-damaging agents, we investigated
their responses to inhibitors of cysteine aspartases, elements of
apoptotic pathways. The V-ICEinh and BAF, two peptide
inhibitors of cysteine aspartases, protected neurons in all three death
paradigms. In contrast, the cysteine aspartase inhibitory peptide
zVAD-fmk conferred protection from NGF withdrawal and SOD1 depletion,
but not DNA-damaging agents, whereas acYVAD-cmk protected only from
SOD1 depletion. Taken together, these findings indicate that three
different apoptotic stimuli activate separate pathways of death in the
same neuron type.
Key words:
CDK; cell cycle; DNA damage; caspase; apoptosis; cytosine
arabinoside
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INTRODUCTION |
Apoptotic death of neurons, even of
the same type of neuron, can be evoked by different initiating events.
For instance, the death of sympathetic neurons can be caused by trophic
factor deprivation (Levi-Montalcini and Angeletti, 1963 ),
peroxynitrite-dependent oxidative stress (Troy and Shelanski, 1994;
Troy et al., 1997), and exposure to DNA-damaging agents (Martin et al.,
1990; Tomkins et al., 1994; Morris and Geller, 1996; Park et al.,
1997).
The capacity of different initiating events to cause apoptotic
death in the same type of neuron raises several issues, including (1)
the extent to which the pathways leading to apoptosis in each case are
distinct and (2) the degree to which they share common elements. Two
specific elements of neuronal apoptotic pathways will be considered
here. First, multiple lines of evidence suggest that cell cycle
components are involved in neuronal death. Several agents that inhibit
cell cycle progression promote survival of trophic factor-deprived
sympathetic neurons and/or neuronally differentiated PC12 cells (Rydel
and Greene; 1988; Rukenstein et al., 1991; Ferrari and Greene, 1994;
Farinelli and Greene, 1996). Neuronal apoptosis also is accompanied by
changes in cyclin-dependent kinase (CDK) activity and cyclin expression
(Brooks et al., 1993; Freeman et al., 1994; Gao and Zalenka, 1995).
Consistent with this, the CDK inhibitors flavopiridol and olomoucine
promote survival of neuronal PC12 cells and sympathetic neurons
deprived of trophic support (Park et al., 1996a).
Components of the cell cycle machinery also may play a role in neuronal
death evoked by DNA-damaging agents. In support of this, flavopiridol
and olomoucine, as well as several G1/S blockers, rescue sympathetic
and cortical neurons from camptothecin-induced death (Park et al.,
1997). The possible role of cell cycle elements in neuronal death
evoked by oxidative stress, however, has not been examined.
Members of the cysteine aspartase (caspase) family of proteases
represent a second element in neuronal death pathways (Ellis et al.,
1991; Miura et al., 1993; Gagliardini et al., 1994). In neuronal cells,
exposure to an antisense oligonucleotide that diminishes expression of
the caspase Nedd-2/caspase 2 protects sympathetic neurons and PC12
cells from NGF deprivation (Troy et al., 1997). In the case of
oxidative stress, studies with naive PC12 cells have established that,
although death in this paradigm is blocked by the
interleuken-converting enzyme-directed inhibitor acYVAD-cmk, the latter
is ineffective in blocking death caused by trophic factor deprivation
(Troy et al., 1996a). Moreover, the Nedd-2 antisense oligonucleotide
that protects from NGF withdrawal does not protect PC12 cells from
oxidative stress (Troy et al., 1997). Such findings indicate that
different caspases are involved in the pathways that regulate apoptosis
evoked by oxidative stress and NGF deprivation.
Caspase involvement in neuronal death caused by DNA-damaging agents has
been less clear. Although the caspase inhibitor zVAD-fmk protects
sympathetic neurons from NGF withdrawal and oxidative stress (Park et
al., 1996b; Troy et al., 1996a), it does not protect them from the
DNA-damaging agent camptothecin (Park et al., 1997). This not only
indicates a divergence of death pathways but also raises the general
issue of whether caspases play a required role in neuronal death evoked
by DNA damage.
The aim of the present studies has been to test further and to refine
our hypotheses about the pathways by which DNA-damaging agents cause
neuronal apoptosis and to compare these pathways with those invoked by
oxidative stress and NGF deprivation. To eliminate potential cell
type-specific differences in death pathways, we focused our examination
on sympathetic neurons. Here, we report that neuronal death evoked by
the DNA-damaging agents cytosine arabinoside (AraC) or UV irradiation,
but not oxidative stress, is inhibited by the CDK inhibitors
flavopiridol and olomoucine. We also provide evidence that death evoked
by DNA-damaging agents requires the actions of cysteine aspartases but
that these are distinct from those involved in apoptosis caused by
withdrawal of trophic support or oxidative stress.
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MATERIALS AND METHODS |
Materials. Human recombinant NGF was kindly provided
by Genentech (South San Francisco, CA). Flavopiridol (L86-8275 [( )
cis-5,7-dihydroxy-2-(2-chlorophenyl)-8[4-(3-hydroxy-1-methyl)-piperidinyl]-4H-benzopyran-4-one]) was a generous gift from Dr. Peter J. Worland (National Cancer Institute, Bethesda, MD). Olomoucine
[2-(2-hydro)-6-benzylamino-9-methylpurine] and iso-olomoucine were
purchased from LC Laboratories (Woburn, MA). Aphidicolin, mouse NGF,
and anti-mouse NGF antiserum were obtained from Sigma (St. Louis, MO).
zVAD-fluoromethylketone (zVAD-fmk) and BAF
Boc-aspartyl(OMe)-fluoromethylketone were purchased from Enzyme Systems
Products (Dublin, CA). The ICEinh peptide (IQACRG; Chiron,
San Diego, CA) and ICEs peptide control (ICGRQA; Chiron) and ASOD1 antisense oligonucleotide were linked to the antennapedia delivery peptide (V; Oncor, Gaithersburg, MD) as previously described (Troy et al., 1996a).
Culture and survival assay of PC12 cells. Naive PC12 cells
were cultured and passaged as previously described (Greene and Tischler, 1976). Neuronally differentiated PC12 cells were generated by
exposing PC12 cells to NGF in serum-free RPMI 1640 medium for 8-9 d.
For survival experiments neuronally differentiated PC12 cells were
plated onto collagen-coated 24-well tissue culture dishes at a density
of ~2 × 105 cells per well. Neuronally
differentiated PC12 cells were cultured in serum-free RPMI 1640 medium
containing NGF (100 ng/ml) throughout the course of survival
experiments. At appropriate times of culture under the conditions
described in the text, cells were lysed, and the numbers of viable
cells were evaluated as previously described (Rukenstein et al., 1991).
All experimental points are expressed as a percentage of cells plated
on day 0 and are reported as mean ± SEM (n = 3).
UV irradiation. Cells were exposed to UV irradiation by
using the Stratolinker UV crosslinker (Stratagene, La Jolla, CA). Sympathetic neurons were exposed to 300 J/m2,
whereas neuronal PC12 cells were exposed to 650 J/m2. Each well was exposed with 200 µl of medium
containing NGF with or without the appropriate drug. After irradiation
300 µl of additional medium containing NGF with or without drug was
added.
Culture and survival assay of rat sympathetic neurons.
Primary cultures of rat sympathetic neurons were generated from
dissociated superior cervical ganglia of postnatal day 1 rats (strain,
Sprague Dawley), as described previously (Lee et al., 1980). The cells were plated onto collagen-coated 24-well dishes at a density of ~0.5
ganglia per well and maintained in RPMI 1640 medium supplemented with
10% heat-inactivated horse serum and 60 ng/ml mouse NGF for a period
of 3 d before the survival experiment. A mixture of uridine and
5-fluorodeoxyuridine (10 µM each) also was added to
eliminate non-neuronal cells on day 2. No pretreatment with a survival
agent, unless otherwise noted, was necessary for effective survival of neurons exposed to AraC. In the case of UV irradiation, the neurons died quite rapidly. Accordingly, neuronal cultures were pretreated overnight (18 hr) with survival-promoting agents before UV irradiation. NGF deprivation was performed by washing with NGF-free medium and the
addition of anti-NGF antiserum, as previously described (Park et al.,
1996a). V-ASOD1 oligonucleotides, in conjunction with the nitric oxide
generator S-nitrosopenicillamine (SNAP; 100 µM), were added to the cultured sympathetic neurons as
previously described (Troy et al., 1997). At appropriate times the
numbers of viable phase-bright neurons were determined by strip
counting, as previously described (Rydel and Greene; 1988). All
experimental points are expressed relative to the original number of
neurons present in each well and are reported as mean ± SEM
(n = 3).
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RESULTS |
The CDK inhibitors flavopiridol and olomoucine promote survival of
sympathetic neurons exposed to UV irradiation or treated with AraC
Our previous studies demonstrated that the camptothecin-induced
apoptotic death of neurons is inhibited by G1/S-phase cell cycle
blockers and CDK inhibitors (Park et al., 1997a), and it was postulated
that camptothecin-induced neurotoxicity is attributable to
transcriptionally mediated DNA strand break formation (Morris and
Geller, 1996) and consequent cell cycle signaling components (Park et
al., 1997).
To assess whether this observation could be extended to two additional
potential DNA-damaging agents, AraC and UV irradiation, we first
determined whether the CDK inhibitors flavopiridol and olomoucine could
prevent the death of cultured sympathetic neurons exposed to UV
irradiation in the presence of NGF. Flavopiridol, a flavonoid
derivative, potently inhibits CDK1/2/ and 4 activities (Losiewicz et
al., 1994; Filgueira de Azevedo et al., 1996) and displays poor
inhibitory activities toward all other kinases examined, including
cAMP-dependent kinase, epidermal growth factor receptor kinase, and
protein kinase C. Olomoucine, a purine derivative, specifically blocks
CDK1/2/ and 5 as well as ERK-1/MAP-kinase activities and was
ineffective against >30 other kinases examined (Vesely et al., 1994).
Both drugs block progression from the G1 to S- and G2 to M-phases of
the cell cycle (Kaur et al., 1992; Vesely et al., 1994). As shown in
Figure 1, both flavopiridol and
olomoucine effectively promoted the survival of UV-treated sympathetic
neurons. At 4 d after UV treatment, ~75-80% of the neurons
were alive with drug treatment, whereas only 25% were alive without
the inhibitors. Maximal protection was observed with 1-3
µM flavopiridol (Fig. 1B) and 200 µM olomoucine (Fig. 1C). These are also the
minimum concentrations required to fully inhibit DNA synthesis by
proliferating PC12 cells (Park et al., 1996a). In addition, the
doses-responses for protection against UV-induced death are nearly
identical to those that rescue sympathetic neurons from both
camptothecin treatment (Park et al., 1997) and NGF deprivation (Park et
al., 1996a). Figure 2 shows the
morphology of sympathetic neurons exposed to UV irradiation in the
presence or absence of flavopiridol or olomoucine. Neurons treated with the CDK inhibitors/UV have a morphology comparable to non-UV-treated control cells (phase-bright cell bodies and intact neurites), whereas
cells treated with UV alone show degenerating cell bodies and neurites.
Iso-olomoucine, an analog control of olomoucine that differs in the
location of one substituent methyl group and that poorly inhibits CDK
activity or DNA synthesis (Park et al., 1996a), was nearly ineffective
in promoting the survival of UV-treated sympathetic neurons (Fig.
1D). The UV treatment experiments were extended to
postmitotic, neuronally differentiated PC12 cells cultured in the
presence of NGF, and similar survival-promoting effects were observed
with olomoucine and flavopiridol (data no shown).
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Figure 1.
The CDK inhibitors, flavopiridol and olomoucine,
inhibit the UV irradiation-induced death of rat sympathetic neurons.
Primary cultures of neonatal rat superior cervical ganglion neurons
were grown in the presence of NGF for 3 d before UV exposure, as
indicated. Neurons were pretreated with the indicated drugs for 16 hr
before UV irradiation. Each data point is the mean ± SEM (n = 3) and is expressed relative to the
number of neurons present in each culture at the time of drug
treatment. p values derived from Student's t test in comparison of the survival of UV-treated and
UV/experimental agent-treated neurons at day 2 are listed.
A, Effects of flavopiridol (1 µM;
p < 0.025) and olomoucine (200 µM;
p < 0.005) on the time course of survival of
sympathetic neurons exposed to UV irradiation. B,
Effects of various doses of flavopiridol on the survival of UV-treated
sympathetic neurons at day 1. C, Effects of various doses of olomoucine on the survival of UV-treated sympathetic neurons
at day 1. D, Effect of iso-olomoucine (200 µM; p > 0.05) on the time course of
survival of sympathetic neurons exposed to UV irradiation.
E, Effect of aphidicolin (10 µM) on the
time course of survival of sympathetic neurons exposed to UV
irradiation.
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Figure 2.
Phase-contrast micrographs of primary sympathetic
neurons maintained in medium containing NGF and treated with the
following: A, UV; B, no treatment;
C, UV + 1 µM flavopiridol;
D, UV + 200 µM olomoucine. Pictures are of
cells at 1 d post-UV exposure.
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The second genotoxic agent we tested was the antitumor drug AraC.
Although the toxic actions of AraC on dividing cells have been
postulated to be attributable to the blockade of DNA synthesis by chain
termination (Ohno et al., 1988), this agent causes neuropathies and
promotes the death of cultured postmitotic sympathetic neurons even in
the presence of NGF (Martin et al., 1990; Tomkins et al., 1994). To
account for the death-promoting actions of AraC on neurons, Martin et
al. (1990) have suggested that it specifically interferes with the NGF
signaling mechanism. In contrast, Tomkins et al. (1994) have proposed
that AraC causes neuronal apoptosis by creating double-stranded breaks
in DNA. Accordingly, we next determined whether flavopiridol and
olomoucine also could inhibit the death of sympathetic neurons. As
Figure 3 shows, both drugs quite
effectively promoted long-term survival of AraC-treated sympathetic
neurons. Approximately 80-100% of the treated neurons survived up to
5 d of AraC exposure, whereas almost all of the neurons in the
control cultures were dead by this time. Beyond this time, toxicity of the CDK inhibitors was observed, even without AraC treatment (Fig. 3A,C). As in the cases of UV-irradiated sympathetic neurons
and neuronal PC12 cells, the maximal concentrations of flavopiridol and
olomoucine required to protect sympathetic neurons from AraC correlated
with the minimum concentrations required to fully inhibit DNA synthesis
by proliferating PC12 cells [1 µM for flavopiridol (Fig.
3B) and 200 µM for olomoucine (Fig.
3D)]. The control compound iso-olomoucine was ineffective
in promoting survival (Fig. 3C). Neurons treated with AraC
and the CDK inhibitors showed the phase-bright morphology of viable
neurite-bearing cells, whereas cells treated with AraC formed
ghost-like cell bodies and degenerating processes (Fig.
4). These results, along with those made
previously (Park et al., 1997), demonstrate that neuronal death induced
by three different forms of DNA-damaging conditions, namely UV
irradiation, camptothecin, and AraC treatment, are blocked effectively
by inhibitors of CDK activity.
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Figure 3.
The CDK inhibitors, flavopiridol and olomoucine,
inhibit AraC-induced death of rat sympathetic neurons. Primary cultures
of neonatal rat superior cervical ganglion neurons were grown in the
presence of NGF for 3 d before drug treatment. Replicate cultures were treated with AraC (100 µM), as indicated. Each
data point is the mean ± SEM
(n = 3) and is expressed relative to the number of
neurons present in each culture at the time of drug treatment. p values derived from Student's t test
in comparison of the survival of AraC-treated and AraC/experimental
agent-treated neurons at day 4 are listed. A, Effect of
flavopiridol (1 µM; p < 0.005) on
the time course of survival of sympathetic neurons treated with AraC.
B, Effect of various doses of flavopiridol on the
survival of AraC-treated sympathetic neurons at day 3. C, Effects of olomoucine (200 µM;
p < 0.005) and iso-olomoucine (200 µM; p > 0.05) on the time course of
survival of sympathetic neurons treated with AraC. D,
Effect of various doses of olomoucine on the survival of AraC-treated sympathetic neurons at day 3. E, Effect of aphidicolin
(10 µM; p > 0.05) on the time course
of survival of sympathetic neurons treated with AraC.
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Figure 4.
Phase-contrast micrographs of primary sympathetic
neurons maintained in medium containing NGF and treated with the
following: A, 100 µM AraC;
B, no treatment; C, AraC + 1 µM flavopiridol; D, AraC + 200 µM olomoucine. Pictures are of cells at 3 d after AraC treatment.
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The S-phase blocker aphidicolin does not promote survival of
sympathetic neurons exposed to UV irradiation or AraC
Previous studies demonstrated that neurons deprived of NGF or
exposed to camptothecin cannot be rescued by cell cycle blocking agents
that act beyond the G1/S border (Farinelli and Greene, 1996; Park et
al., 1997). For example, the S-phase blocker aphidicolin is ineffective
in blocking the death of sympathetic neurons in both paradigms
(Farinelli and Greene, 1996; Park et al., 1997). Consistent with this,
aphidicolin also failed to block the death of neurons exposed either to
UV irradiation (see Fig. 1E) or to AraC (Fig.
3E). Such findings demonstrate that inhibition of DNA synthesis per se does not promote survival of neurons induced to die by
UV, AraC, camptothecin, or NGF deprivation.
The CDK inhibitors flavopiridol and olomoucine do not promote
survival of sympathetic neurons subjected to oxidative stress
Sympathetic neurons depleted of SOD1 by treatment with an SOD1
antisense oligonucleotide and exposed to nitric oxide-generating compounds die apoptotically, apparently because of formation of peroxynitrite (Troy et al., 1996b, 1997). We examined whether the CDK
inhibitors would block the death of neurons induced by this means. As
shown in Figure 5, neither flavopiridol
nor olomoucine protected sympathetic neurons in this paradigm of
oxidative stress. However, in the same experiments, and as anticipated
from previous studies (Park et al., 1996a), both inhibitors effectively
blocked the death of sympathetic neurons deprived of NGF. This suggests that cell cycle regulatory components do not control the neuronal death
pathways induced by oxidative stress. The CDK inhibitors also failed to
inhibit the death of neuronal PC12 cells depleted of SOD1 (data not
shown).
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Figure 5.
The CDK inhibitors, flavopiridol
(FL; 3 µM) and olomoucine
(OL; 200 µM), do not inhibit the death of
sympathetic neurons depleted of SOD1. Primary cultures of neonatal rat
superior cervical ganglion neurons were grown in the presence of NGF
for 3 d before drug treatment. Replicate cultures were deprived of
NGF (antiNGF) or treated with ASOD1 (50 nM)/SNAP (100 µM; referred to as
ASOD1 in the figure), as indicated, for 3 d. Each
data point is the mean ± SEM
(n = 3) and is expressed relative to the number of neurons present in each culture at the time of drug treatment. p values derived from Student's t test
are p < 0.005 for anti-NGF versus anti-NGF/CDK
inhibitors and p > 0.05 for ASOD1 versus ASOD1/CDK inhibitors.
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Differential effects of cysteine aspartase inhibitors on
sympathetic neurons deprived of NGF, depleted of SOD1, or exposed to
DNA-damaging agents
We next compared the role of cysteine aspartases in our three
paradigms for inducing apoptotic neuronal death. As shown in Figure
6, zVAD-fmk (50 µM), an
irreversible peptide inhibitor of several different cysteine aspartases
(Stefanis et al., 1996; Livingston, 1997), increases the survival of
sympathetic neurons from death evoked by oxidative stress (via exposure
to v-ASOD1/nitric oxide generators). This inhibitor also was found to
protect PC12 cells from apoptosis caused by SOD1 depletion (Troy et
al., 1996a). Additionally, zVAD-fmk (50 µM) protected
sympathetic neurons from NGF deprivation (Fig. 6A).
In contrast to its effects on sympathetic neurons subjected to
oxidative stress or NGF deprivation, 50 µM zVAD-fmk was
ineffective in promoting survival of these neurons after exposure to UV
irradiation (Fig. 6A) or AraC (Fig.
6B). We previously reported similar findings for
exposure to camptothecin (Park et al., 1997). Increasing the zVAD-fmk
concentration to 100 µM had minimal effect (data not
shown).
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Figure 6.
The cysteine aspartase family protease inhibitors
zVAD-fmk and acYVAD-cmk differentially promote survival of sympathetic
neurons exposed to DNA-damaging agents, deprived of NGF, or depleted of SOD1. Primary cultures of neonatal rat superior cervical ganglion neurons were grown in the presence of NGF for 3 d
before experimentation. Cultures were deprived of NGF and treated as
indicated for 3 d before assessment of neuronal survival. Neurons
exposed to UV irradiation were pretreated with zVAD-fmk for 16 hr.
p values derived from Student's t test
are included, as indicated. A, Effect of zVAD-fmk (50 µM) on the time course of survival of sympathetic neurons
exposed to UV irradiation (p > 0.05, day 2)
and deprived of NGF (p < 0.005, day 2).
B, Effect of zVAD-fmk (50 µM) on the time
course of survival of sympathetic neurons treated with AraC (100 µM; p > 0.05, day 3).
C, Effect of acYVAD-fmk [250 µM;
p > 0.05 (antiNGF);
p < 0.025 (ASOD1)] and zVAD-fmk
[50 µM; p < 0.005 (antiNGF); p < 0.01 (ASOD1)] on the survival of neurons deprived of NGF
(antiNGF) or treated with antisense to SOD1
(ASOD1), along with SNAP (100 µM).
Survival was measured 2 d after NGF deprivation or treatment with
ASOD1/SNAP. Each data point is the mean ± SEM
(n = 3) and is expressed relative to the number of neurons present in each well at the time of drug treatment.
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The above observations indicate a distinction between the
pathways by which DNA-damaging agents evoke death and by which
oxidative stress and NGF deprivation do so. A previous study showed
that sensitivity to the peptide acYVAD-cmk, a more specific inhibitor of ICE-like proteases than zVAD-fmk, blocks the death of naive PC12
cells caused by oxidative stress but has no effect in the trophic
factor deprivation paradigm (Troy et al., 1996a). Figure 6C
shows that acYVAD-cmk shows similar selectivity between these two
causes of death in sympathetic neurons. Thus, cysteine protease inhibitors distinguish among each of the three causes of sympathetic neuron death studied here.
Our observations raise the question as to whether any cysteine
aspartase is required for death induced by DNA-damaging agents. To
examine this issue, we used two additional cysteine aspartase inhibitors, the antennapedia vector peptide-linked ICEinh
and the irreversible peptide inhibitor BAF. ICEinh is a
peptide (IQACRG) that includes the conserved active site of ICE-family
proteases (Troy et al., 1996a). Because nearly all known cysteine
aspartases possess this conserved sequence, it is expected that
ICEinh would compete with caspases for the binding of
substrates and therefore block apoptotic death. The antennapedia vector
peptide (V) crosses plasma membranes with high efficiency and functions
as a vector to facilitate cellular uptake of the ICEinh
peptide (Troy et al., 1996a). The two peptides are linked by a
reducible S-S bond, and V-ICEinh is cleaved within cells,
releasing free ICEinh, which accumulates
intracellularly (Troy et al., 1996a). A previous report demonstrated
that V-ICEinh blocks the death of PC 12 cells and sympathetic neurons deprived of trophic support (Troy et al., 1996a).
In addition, it has been shown to inhibit the death of PC12 cells
depleted of SOD1 (Troy et al., 1996a). Figure
7 shows that, under conditions in which
V-ICEinh protects sympathetic neurons from NGF deprivation,
it also blocks the death of sympathetic neurons exposed to UV (~80%
survival with the inhibitor vs 25% without V-ICEinh
treatment; Fig. 7A) or treated with AraC (~90% survival
with V-ICEinh vs 50% without inhibitor treatment on day 2;
Fig. 7B). V-ICEinh additionally blocked the
death of sympathetic neurons either exposed to camptothecin or
subjected to SOD1 depletion/NO generators (data not shown). A scrambled
peptide (ICGRQA) linked to antennapedia (termed
V-ICEs) was used as a control for nonspecific effects and was ineffective in promoting survival of neurons in all
paradigms of death (Fig. 7; Troy et al., 1996a).
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Figure 7.
The cysteine aspartase family protease inhibitor
V-ICEinh protects sympathetic neurons from NGF deprivation
and UV- and AraC-induced apoptosis. Primary cultures of neonatal rat
superior cervical ganglion neurons were grown in the presence of NGF
for 3 d before experimentation. On the third day neurons were
pretreated for 18 hr in the presence of NGF with the
V-ICEinh inhibitor (200 nM) or the
V-ICEs control (200 nM), as indicated. Then
neurons were exposed to UV irradiation or AraC (100 µM)
or were deprived of NGF. p values derived from
Student's t test are included as indicated.
A, Effect of V-ICEinh inhibitor (200 nM; p < 0.005) and V-ICEs
(200 nM; p > 0.05) on the survival of
sympathetic neurons exposed to UV irradiation. B, Effect of V-ICEinh inhibitor (200 nM; p < 0.025, day 2) and
V-ICEs (200 nM; p > 0.05, day 2) on the survival of sympathetic neurons exposed to AraC.
C, Effect of V-ICEinh inhibitor (200 nM; p < 0.01) and V-ICEs
(200 nM; p > 0.05) on the survival of
sympathetic neurons deprived of NGF. Each data point is
the mean ± SEM (n = 3) and is expressed
relative to the number of neurons present in each well at the time of
drug treatment.
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BAF is a cell-permeant irreversible cysteine aspartase inhibitor
that blocks the death of Fas-treated thymocytes and NGF-deprived sympathetic neurons (Deshmukh et al., 1996). Consistent with the protective effect of V-ICEinh, BAF also reduces the
death of sympathetic neurons treated with all three DNA-damaging
agents. Treatment with 100 µM BAF fully protected
sympathetic neurons from AraC-induced death at day 3, whereas 75%
death was observed in control cultures (Fig.
8A). In addition, BAF
protected sympathetic neurons from UV irradiation (~80% survival
with BAF treatment vs <40% without; Fig. 8B).
Similar protective effects of BAF were observed with camptothecin-treated sympathetic neurons (data not shown). The IC50 of BAF for prevention of death in all three paradigms
of DNA damage was ~25 µM. As previously reported
(Deshmukh et al., 1996), BAF treatment totally protected sympathetic
neurons from death induced by NGF deprivation (Fig. 8C). BAF
treatment alone had no toxic effect on neuronal survival (data not
shown). Taken together, these data indicate that the death pathways
evoked by DNA-damaging agents require the activation of one or more
cysteine aspartase(s), but that these are different from those that act in the pathways triggered by NGF deprivation and SOD1 depletion.
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Figure 8.
The cysteine aspartase family protease inhibitor
BAF protects sympathetic neurons from NGF deprivation and UV- and
AraC-induced apoptosis. Primary cultures of neonatal rat superior
cervical ganglion neurons were grown in the presence of NGF for 3 d before experimentation. Cultures were deprived of NGF and treated as indicated for 3 d before assessment of neuronal survival. Neurons exposed to UV irradiation were pretreated with BAF for 16 hr. p values derived from Student's t test
are included as indicated. A, Effect of BAF (100 µM; p < 0.005, day 2) on the time
course of survival of sympathetic neurons exposed to UV irradiation. B, Effect of BAF (100 µM;
p < 0.025, day 3) on the time course of survival
of sympathetic neurons treated with AraC (100 µM). C, Effect of BAF (100 µM;
p < 0.025, day 2) on the time course of survival
of neurons deprived of NGF. Each data point is the mean ± SEM (n = 3) and is expressed relative
to the number of neurons present in each well at the time of drug
treatment.
|
|
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DISCUSSION |
In the present studies we explored the signaling pathways by
which DNA-damaging agents cause the death of postmitotic neurons and
compared such mechanisms with those involved in neuronal apoptosis caused by the loss of trophic support and oxidative stress.
Involvement of cell cycle signals in neuronal
death caused by DNA-damaging agents
Our studies considered three conditions that damage DNA and
trigger apoptotic death: UV irradiation, camptothecin, and AraC treatment. UV irradiation produces direct DNA damage that is
mediated primarily by the formation of pyrimidine dimers (Schreiber et al., 1995). Camptothecin binds to and inhibits DNA topoisomerase I,
forming a cleavable complex consisting of topo-I covalently linked to
the severed DNA strand (Hsiang et al., 1989). In postmitotic neurons it
is postulated that the transcription machinery collides with this
complex, resulting in a lethal DNA lesion (Morris and Geller, 1996).
The chain-terminating nucleoside, AraC, also kills postmitotic neurons.
However, the mechanism of AraC neurotoxicity currently remains unclear.
Tomkins et al. (1994) have suggested that AraC-induced death of
sympathetic neurons could be attributable to either inhibition of
topo-II-mediated DNA repair or inhibition of DNA ligase, both of which
would lead to the persistent formation of DNA strand breaks.
How might DNA damage lead to neuronal death? At least for proliferating
cells, a growing body of evidence suggests the involvement of cell
cycle-related components. For instance, the cell cycle-related tumor
suppressor protein p53 appears to mediate the responses of many cell
types to UV-evoked DNA damage (Sanchez and Elledge, 1995). It appears
that p53 expression also is required for AraC-evoked death of neurons
(Enokido et al., 1996). Furthermore, AraC treatment has been reported
to increase cyclin E-associated CDK activities in HL 60 cells (Ping Dou
et al., 1995), whereas camptothecin upregulates cdc2 (Shimizu et al.,
1995) and cyclin D1 (Chen et al., 1995) levels in HL60 and RKO cells,
respectively.
Previous evidence supports the hypothesis that components of the cell
cycle machinery contribute to the death of postmitotic neurons caused
by loss of trophic support (Ferrari and Greene, 1994; Farinelli and
Greene, 1996; Park et al., 1996a). Here, we showed that the CDK
inhibitors flavopiridol and olomoucine effectively suppress neuronal
death caused by Ara C and UV treatment. Moreover, as with camptothecin
(Park et al., 1997), the concentrations of CDK inhibitors required to
block death correlate well with the levels required to inhibit DNA
synthesis in proliferating PC12 cells. This extends our findings to two
additional treatments that are quite distinct from one another and from
camptothecin with respect to the initial mechanism but that also have
the capacity to cause DNA damage. Such observations support the
hypotheses (1) that DNA strand breaks are a common element in the
pathways by which these treatments cause neuronal apoptosis and (2)
that death in each instance involves components of the cell cycle
machinery. For all three DNA-damaging agents, aphidicolin, which blocks
S-phase by inhibiting DNA polymerase  , was ineffective in providing
protection. This suggests that inhibition of active DNA synthesis per
se is not sufficient to promote survival.
Interestingly, flavopiridol and olomoucine are cytotoxic to numerous
oncogenic cell lines, including dividing PC12 cells (Bible and
Kaufmann, 1996; Park et al., 1996a). Although the exact mechanisms by
which these CDK inhibitors kill transformed cells are unknown, one
interpretation is that death occurs as a result of a signaling conflict
brought on by inhibition of CDK activity and constitutively present
oncogenic signal(s). Presumably, in the case of neuronal PC12 cells,
this conflict is avoided because of the downregulation of
oncogenic/proliferative signals during differentiation. Such oncogenic/proliferative signals also would be absent in neurons, and
these too are therefore resistant to the cytotoxic effects of the CDK
inhibitors.
Although our findings with olomoucine and
flavopiridol suggest the involvement of CDKs in the pathway by which
DNA-damaging agents lead to neuronal apoptosis, alternative
interpretations must be considered. In particular, we cannot rule out
entirely the possibility that flavopiridol and olomoucine act on
required elements in death signaling other than CDKs. We previously
have eliminated effects of these drugs on JNK activity or activation (Park et al., 1996a), which appear to be required for the death of
NGF-deprived neuronally differentiated PC12 cells (Xia et al., 1996).
In addition, it was shown previously that attenuation of ERK activation
does not affect survival of neuronal cells, suggesting that olomoucine
does not act to promote survival via inhibition of ERK activity (Park
et al., 1996a; Virdee and Tolkovsky, 1996). Although all our findings
to date as well as those of others (Losiewicz et al., 1994; Vesely et
al., 1994) have pointed to the highly selective nature of these CDK
inhibitors, it remains conceivable that they affect kinases yet to be
examined that play a required role in cell death.
CDK inhibitors do not protect neurons from oxidative stress
Depletion of SOD1 in the case of PC12 cells, and SOD1
depletion combined with exposure to NO generators in the case of
sympathetic neurons, leads to apoptotic death by what appears to be a
peroxynitrite-dependent mechanism (Troy et al., 1996b). Evidence has
been presented for the role of peroxynitrite in neurodegenerative
disorders, and so the mechanism by which this agent results in
apoptosis is of both basic and clinical relevance (Coyle and
Puttfarcken, 1993). Because peroxynitrite can lead to cellular DNA
damage, we considered the possibility that, like DNA-damaging agents,
it might evoke death by a mechanism dependent on cell cycle regulatory
components. However, in contrast to our observations with NGF
deprivation and DNA-damaging agents, death in the SOD1 depletion
paradigm was insensitive to flavopiridol and olomoucine. This suggests that CDKs do not regulate the death of sympathetic neurons in all
paradigms of neuronal death.
Different cysteine aspartases mediate the death of neurons deprived
of NGF, depleted of SOD1, or exposed to DNA-damaging conditions
Although our findings suggest that CDK activity mediates some part
of the apoptotic signaling response in the case of trophic factor
deprivation and DNA damage, it is not clear to what extent the
signaling pathways activated by these two apoptotic stimuli are
identical and to what extent they may differ. In addition, a notable
feature of our experiments was that AraC, UV irradiation, and
camptothecin each evoked apoptosis in the presence of NGF. Accordingly,
an alternative interpretation, as has been suggested for AraC (Martin
et al., 1990), is that all or some of the apoptosis-evoking conditions
we used work by blocking the signaling pathway by which NGF promotes
survival. To address these issues, we examined whether the death of
sympathetic neurons caused by different stimuli can be blocked by
inhibitors of cysteine aspartases. The cysteine aspartase inhibitor
zVAD-fmk blocked death evoked by NGF deprivation and SOD1 depletion,
but not by AraC or UV irradiation. We previously reported similar
findings for camptothecin (Park et al., 1997a). In addition, we show
here that apoptosis in response to NGF deprivation and oxidative stress
in sympathetic neurons, as in PC12 cells (Troy et al., 1996a), also
differ in their response to caspase inhibitors. acYVAD-cmk, an
inhibitor more specific to the ICE-like caspases, blocks death evoked
by SOD1 depletion, but not death caused by NGF deprivation. These
observations indicate that the death pathways triggered by NGF
deprivation, SOD1 depletion, and DNA-damaging agents are distinct from
one another. They also rule out the action of DNA-damaging agents,
including AraC, as inhibitors of proximal elements of the NGF signaling
mechanism.
Our observations suggest that death elicited by NGF
withdrawal and DNA-damaging agents might involve different members of the cysteine protease family, because death caused by DNA-damaging agents is insensitive to zVAD-fmk. It must be noted that the exact nature of the inhibitory efficacies and potencies of zVAD-fmk on
various known members of the cysteine aspartase family has yet to be
reported. Although we have shown that zVAD-fmk can block the processing
of IL-I by ICE (L. Stefanis, unpublished results) and cleavage of a
fluorogenic DEVD substrate by a CPP32-like activity in cell extracts
taken from trophic factor-deprived PC12 cells (Stefanis et al., 1996),
others have reported that zVAD-fmk is not a potent inhibitor of
PARP-cleaving protease activity in THP.1 cells (Slee et al., 1996).
This suggests that zVAD-fmk is not a universal inhibitor of cysteine
aspartases. To address whether different caspases might mediate the
death of sympathetic neurons exposed to differential apoptotic stimuli,
we tested other known caspase inhibitors. BAF has been shown previously
to inhibit ICE and CPP32 in vitro, but not calpain I or II,
indicating that it acts as an inhibitor of cysteine aspartases, but not
other cysteine proteases (Deshmukh et al., 1996). In addition, it was
reported to block the death of Fas-treated thymocytes and NGF-deprived sympathetic neurons (Deshmukh et al., 1996). Similarly,
V-ICEinh mimics the active site of almost all known
cysteine aspartase family members and should function as a general
inhibitor of these enzymes (Troy et al., 1996a). We show here that both
BAF and V-ICEinh inhibit the death of sympathetic neurons
evoked by all three DNA-damaging agents and NGF deprivation,
implicating the action of one or more caspases. Taken together, these
data indicate that apoptosis caused by DNA-damaging agents is dependent
on one or more cysteine aspartase family members and that the identity
of this/these is different from the family member(s) that mediate
apoptosis caused by NGF withdrawal and oxidative stress.
The identity of the cysteine aspartase(s) required for neuronal death
caused by DNA-damaging agents presently is unknown. Previous work
compared the cysteine aspartases that mediate the death of PC12 cells
and sympathetic neurons caused by either NGF deprivation or
downregulation of SOD1 (Troy et al., 1996a). Although death in both
cases was inhibited by V-ICEinh and zVAD-fmk, antisense oligonucleotides to Nedd-2 rescued PC12 cells and sympathetic neurons
from withdrawal of NGF, but not from SOD1 downregulation (Troy et al.,
1996a, 1997). Conversely, acYVAD-cmk, as well as blocking antibodies to
mature IL-1 and an IL-1 receptor antagonist, protected PC12 cells
from SOD1 depletion, but not NGF withdrawal (Troy et al., 1996a). Our
experiments extend the observations with acYVAD-cmk to sympathetic
neurons and thus lend further support to the model that distinct
caspase-dependent pathways mediate neuronal apoptosis evoked by NGF
withdrawal (Nedd-2 dependent) and SOD1 depletion (ICE-like
dependent).
In summary, we report that NGF deprivation, DNA-damaging agents, and
oxidative stress activate different apoptotic pathways in sympathetic
neurons. The properties of these pathways are summarized in Figure
9. As shown in this figure, past studies
have placed cell cycle signals upstream of caspase activation (Stefanis
et al., 1996). For oxidative stress, formation of peroxynitrite appears to be the initiating cause of death, and therefore this also lies upstream of caspase activation (Troy et al., 1996b). In the cases of
NGF deprivation and DNA damage-induced neuronal death, there is
evidence for the involvement of CDKs, whereas this is not the case for
the oxidative stress model. In addition, each of the three pathways of
death uses distinct cysteine aspartases. Thus, even in the
same neuron type, multiple apoptotic pathways can be
activated, depending on the initiating stimulus of death.
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Figure 9.
Model for the differential death pathways induced
by trophic factor deprivation, DNA damage, and superoxide dismutase 1 reduction in sympathetic neurons.
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FOOTNOTES |
Received Sept. 4, 1997; revised Oct. 23, 1997; accepted Nov. 10, 1997.
This work was supported in part by Grants from National Institutes of
Health (NS33689), March of Dimes, Blanchette Rockefeller Foundation,
Amyotrophic Lateral Sclerosis Foundation, and the Aaron Diamond
Foundation (to L.A.G.); by Grants NS36443 and ES06897 from National
Institutes of Health and grants from the Cancer Institute of New Jersey
(to H.M.G.) and from Muscular Dystrophy Association (to C.M.T.). DSP is
an Aaron Diamond Foundation Fellow. We thank Dr. Peter J. Worland for
kindly donating flavopiridol and for his helpful discussions.
Correspondence should be addressed to Dr. David S. Park, Department of
Pathology, Taub Center for Alzheimer's Disease Research, Columbia
University College of Physicians and Surgeons, 630 West 168th Street,
New York, NY 10032.
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Top
Abstract
Introduction
