From the Institute for Brain Aging and Dementia and
the ¶ Department of Developmental and Cell Biology, University of
California at Irvine, Irvine, California 92697
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ABSTRACT |
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Stable transfectants of PC12 cells expressing
bcl-2 or crmA were generated and tested for their susceptibility to
various apoptotic insults. Bcl-2 expression conferred resistance to
apoptosis induced by staurosporine and by oxidative insults including
hydrogen peroxide and peroxynitrite, but was less effective in
inhibition of activation-induced programmed cell death induced by
concanavalin A. Concanavalin A-induced apoptosis was abated, however,
in cells expressing very high levels of bcl-2. In contrast, cells
expressing crmA were protected from concanavalin A-induced apoptosis,
but were as susceptible as control cells to apoptosis induced by
staurosporine and oxidative insults. Therefore, at least two apoptotic
pathways in PC12 cells can be discerned by their differential
sensitivity to blockade by bcl-2 and crmA. The ability of Alzheimer's disease
(AD)1 is characterized by a
pronounced loss of neurons in susceptible regions of the brain (1).
Evidence suggests that this neuronal loss occurs through apoptosis (2, 3), a type of cell death with distinct morphological and biochemical characteristics. The principle component of senile plaques in AD brain
is A Recent studies have demonstrated that multiple pathways of apoptosis
can be discerned by their differential sensitivity to blockade. In one
model of APCD, Fas ligand binds to its receptor, causing receptor
aggregation, recruitment of death domain- and death effector
domain-containing proteins, and activation of a cascade of caspase
proteases (16). APCD induced by Fas can be blocked by the viral serpin
crmA (17), a caspase inhibitor whose physiological target is likely to
be the apical caspase in the cascade (18), but it is insensitive to
blockade by bcl-2 (Refs. 19-21, but also see Refs. 22-24). In
contrast, apoptosis induced by the protein kinase inhibitor
staurosporine (STS) is not inhibited by crmA (25), but is very
efficiently blocked by bcl-2 (26). Other insults initiate apoptosis
which is similarly sensitive to blockade by bcl-2 but insensitive to
crmA, including etoposide, a DNA topoisomerase inhibitor (27).
In order to differentiate pathways used by apoptotic insults, stable
transfectant PC12 cell lines expressing crmA or bcl-2 were generated.
The results shown here demonstrate that APCD and non-APCD pathways of
apoptosis differentially sensitive to inhibition by crmA and bcl-2
exist in PC12 cells. APCD induced by concanavalin A (ConA) is blocked
by crmA, while apoptosis elicited by STS and oxidative insults is
blocked by bcl-2. Importantly, A Vectors--
The complete coding sequence of human bcl-2
(provided by D. Hockenbery; Fred Hutchinson Cancer Research Center,
Seattle, WA) (28) was subcloned into the pCDNA3 expression vector
(Invitrogen) which uses the cytomegalovirus promoter to direct high
levels of transgene expression in many types of cells. Similarly, a
cDNA encoding the viral serpin crmA (provided by D. J. Pickup,
Duke University) (29) was subcloned into pCDNA3. An IRES (internal ribosome entry site)-lacZ reporter sequence (30) was subcloned into the
pHSVpuc amplicon (generously provided by F. Lim, Universidad Autonoma
de Madrid). CrmA or bcl-2 cDNAs were subcloned upstream of the
IRES-lacZ sequence to generate amplicons directing expression of
bicistronic mRNAs.
PC12 Cell Culture--
The PC6 subline of PC12 cells obtained
from R. N. Pittman (University of Pennsylvania) (31) was grown in
DMEM containing 10% horse serum, 5% fetal calf serum, and
penicillin/streptomycin in 5% CO2. Cells were transfected
with bcl-2/pcDNA3, crmA/pcDNA3, or pcDNA3 (the vector
control) using LipofectAMINE (Life Technologies, Inc.) and stable
transfectants were selected with 0.5 mg/ml G418. Several independent
clones were isolated, recloned, and selected for use.
Drugs--
For assays using PC12 cells, a 1 mM stock
solution of STS (Calbiochem) was made in dimethyl sulfoxide. ConA
(Sigma) was dissolved in Opti-MEM. Hydrogen peroxide (37%, Sigma) was
diluted in Opti-MEM immediately before use. Peroxynitrite (Upstate
Biotechnology) was used at 100 µM. A 1.32 mM
stock solution of FeSO4 was made in Opti-MEM.
A PC12 Cell Toxicity Assays--
Cells were passaged at 1e4
cells/cm2 into 48-well tissue culture dishes coated with 50 µg/ml poly-D-lysine and 20 µg/ml type 1 collagen
(Sigma). The following day, cells were rinsed and the medium was
replaced with Opti-MEM (Life Technologies, Inc.) supplemented with 2 mM CaCl2. After 4 h, drugs were added, and
24 to 28 h later cell viability was assessed by nuclear
morphology. Nuclei in live cells or cells previously fixed with 4%
paraformaldehyde in phosphate-buffered saline were stained by the
addition of 0.5 µM SYTO11 (Molecular Probes), and cells
were visualized with phase-contrast and epifluorescence microscopy. For
each condition, live/dead cell counts were obtained from 2-3 fields of
3-4 wells. Cells with large nuclei containing uniformly stained
chromatin were counted as live cells, while cells containing fragmented
nuclei and/or condensed chromatin were counted as dead cells. None of
the drugs used caused changes in the total cell number (live + dead)
over the course of the assay. Therefore, the number of live treated
cells, expressed as the percentage of the number of live
vehicle-treated cells, was used as a measurement of cell viability.
Data shown are from representative experiments. Each experiment was
repeated several times with similar results. In some experiments the
ability of cells to oxidize 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) was used as a measurement of cell viability
(32). Similar results were obtained regardless of the method used to assess cell viability when STS and ConA were used to induce cell death.
Western Blots--
Cells were rinsed with Opti-MEM and total
cell lysates were prepared by scraping cells into 1% SDS, 10 mM Tris, pH 7.5, 1 mM EDTA. Protein was
estimated by the BCA assay (Pierce), and 1-15 µg of total cellular
protein was separated by 12% SDS-polyacrylamide gel electrophoresis
and blotted onto Immobilon-P polyvinylidene difluoride (Invitrogen).
Blots were blocked with 3% goat serum, 2% bovine serum albumin in TBS
containing 0.1% Tween 20 and incubated with rabbit anti-bcl-2 (Santa
Cruz 492 at 1:1000) in block. Antibody was detected using horseradish
peroxidase-conjugated secondary antibody and ECL reagent (Amersham).
Films were scanned and analyzed using NIH Image.
Primary Cultures of Hippocampal Neurons--
Hippocampi of E18
rat embryos were dissected in CMF (calcium- and magnesium-free
Hanks'-balanced salt solution, containing 20 mM HEPES, 1 mM pyruvate, 4.2 mM sodium bicarbonate, and
0.3% bovine serum albumin), rinsed with CMF, and resuspended in a
trypsin solution (0.125% trypsin in CMF containing 0.5 mM
EDTA) for 8 min at 37 °C. The trypsinization was stopped by the
addition of DMEM containing 10% fetal calf serum, and the tissue was
centrifuged at 200 × g for 5 min. The resulting cell
pellet was resuspended in 2 ml of culture medium (DMEM, Life
Technologies, Inc., 12100-046, containing 20 mM HEPES, 26.2 mM sodium bicarbonate, 1 mM sodium pyruvate,
and B27 supplement, Life Technologies, Inc.). Following trituration
through fire-polished Pasteur pipettes with the diameter maximally 50%
constricted, the cell suspension was filtered through a 40-µm cell
strainer (Falcon), and viable cells were counted using trypan blue.
Cells were plated at 5-8e4 cells/cm2 in 48-well tissue
culture dishes (Costar).
Transduction of Neurons with Herpes Simplex Virus-1 Amplicons and
Toxicity Assays--
Amplicons were packaged using replication
incompetent 5dl1.2 helper virus (obtained from P. A. Schaffer, University of Pennsylvania) and 2-2 cells (provided by R. Sandri-Goldin, University of California, Irvine) and purified through
sucrose gradient centrifugation as described previously (33). Viral
vectors were titered on PC12 cells as described (33). Hippocampal
neurons were infected at a multiplicity of infection of 0.1-0.01 with
crmA-IRES-lacZ, bcl2-IRES-lacZ, or IRES-lacZ vectors after 2 days in
culture. Cells were treated with drugs the next day and fixed 24 h
later by underlay with 4% paraformaldehyde in phosphate-buffered
saline containing 5% sucrose for 30 min. Fixed cultures were blocked
in TBS containing 0.3% Tween 30, 3% goat serum, and 2% bovine serum
albumin and then incubated with 40-1a (1:4000 ascites)
anti- In preliminary experiments to establish dose-response curves for
cell death, PC12 cells were exposed to increasing concentrations of
STS, ConA, A-amyloid
(A
) to induce apoptosis in these cells was assessed. CrmA
transfectants were protected from apoptosis induced by
A
1-42, but only cells expressing very high levels
of bcl-2 were similarly protected. These results suggest that the
apoptotic pathway activated by A
1-42 in PC12 cells can
be differentiated from the apoptotic pathway activated by oxidative
insults. Gene transfer experiments also demonstrated that expression of
crmA in primary cultures of hippocampal neurons is protective against
cell death induced by A
1-42. Together these results
support the hypothesis that A
-induced apoptosis occurs through
activation-induced programmed cell death.
INTRODUCTION
Top
Abstract
Introduction
References
-amyloid (A
), a 39-43-amino acid peptide derived from amyloid
precursor protein. A
has been shown to be neurotoxic in
vivo (4) and in vitro (5-8), and is generally believed to contribute to the etiology of AD. Understanding how A
induces neuronal apoptosis, therefore, may be important for clinical
interventions in AD.
must aggregate into a
-pleated sheet structure to induce the
death of cultured hippocampal neurons (6), and A
is not toxic when
immobilized as a neuronal substrate (9). These findings have led to the
suggestion that aggregated A
might cross-link transmembrane plasma
membrane receptors to initiate a death program, in a type of
activation-induced programmed cell death (APCD) (10). Studies of A
effects on cells have documented changes in tyrosine phosphorylation of
cellular substrates (11, 12), suggesting that activation of signal
transduction pathways might also be involved. Because A
causes
oxidative stress in neurons, A
has been proposed to cause death
through an oxidative mechanism (13), and antioxidants have sometimes
(13) but not always (14) been reported to block A
-induced cell
death. Other studies have suggested that A
perturbs calcium
homeostasis in neurons to cause cell death (15).
-induced cell death is blocked by
crmA, suggesting that A
may cross-link cell surface receptors to
engage an APCD apoptotic pathway.
EXPERIMENTAL PROCEDURES
1-42 and A
25-35, synthesized and
purified as described previously (6), were obtained from C. Glabe
(University of California, Irvine). A stock solution of
A
1-42 was made in Opti-MEM and used after one
freeze-thaw cycle. A
25-35 was dissolved in
ddH2O at 2.5 mM and used after several
freeze-thaw cycles. zVAD-fmk (Bachem) was dissolved in dimethyl
sulfoxide at 100 mM. Glutathione ethyl ester (GSH) and
propyl gallate (Sigma) were dissolved in Opti-MEM. For assays using
primary cultures of hippocampal neurons, drugs were prepared similarly,
with the exceptions that Opti-MEM was replaced by DMEM-B27 medium, and A
1-42 was dissolved in ddH2O and used after
1 week at 4 °C.
-galactosidase (developed by Joshua Sanes and obtained from the
Developmental Studies Hybridoma Bank maintained by the University of
Iowa Department of Biological Sciences under contract NO1-HD-7-3263
from the National Institute of Child Health and Human Development,
National Institutes of Health).
-Galactosidase immunoreactivity was
detected by incubation with 7 µg/ml cy2-antimouse IgG (Jackson
Immunoresearch Laboratories). Nuclear morphology was assayed by
inclusion of 1.25 µg/ml Hoechst 33258 (Sigma) in the secondary
antibody incubation. Cells were visualized by epifluorescence
microscopy. Noninfected cells and cells infected with 5dl1.2
helper virus were uniformly negative for
-galactosidase
immunoreactivity. Cells transduced by amplicons, identified by the
presence of
-galactosidase immunoreactivity, were counted and their
nuclear morphology (normal or apoptotic) recorded. Results are
presented as the number of infected cells with normal nuclei after drug
treatment as a percentage of the number of infected cells with normal
nuclei after vehicle control treatment.
RESULTS
1-42, A
25-35, or hydrogen
peroxide in Opti-MEM, a low protein-containing chemically defined
medium. Cell viability was assessed after 24 h by nuclear
morphology visualized with the fluorescent dye SYTO11, since this
method allows clear visualization of apoptotic nuclei (Fig.
1). Over 95% of control cells were
viable after serum withdrawal into Opti-MEM, but exposure to these
insults caused dose-dependent apoptotic death of the cells,
evidenced by somal shrinkage, plasma membrane blebbing, chromatin
condensation, and nuclear fragmentation. Drug concentrations that
caused 50-90% of the cells to die over the course of 24 h were
chosen for use in subsequent assays. Preincubation with the cell
permeable caspase inhibitor zVAD-fmk completely blocked cell death
induced by these concentrations of ConA, STS, A
1-42, and A
25-35, and partially blocked cell death induced by this concentration of hydrogen peroxide, consistent with the
involvement of apoptotic pathways in the observed cell death (Fig.
2).
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Fig. 1.
Changes in nuclear morphology produced by
apoptotic insults in PC12 cells. Nuclear morphology in fixed
cultures of PC12 cells was visualized by epifluorescence of the
chromatin dye SYTO11. The nuclei of untreated PC12 cells are large and
chromatin is diffusely stained. Cell death produced by exposure to ConA
(100 nM), STS (250 nM), hydrogen peroxide (150 µM), A 1-42 (50 µM), or
A
25-35 (25 µM) is associated with
chromatin condensation and nuclear fragmentation. Arrowheads
indicate healthy nuclei; arrows indicate condensed or
fragmented nuclei.
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Fig. 2.
Inhibition of cell death by zVAD-fmk.
Cell viability was assessed in PC12 cells pretreated for 2 h with
100 µM zVAD-fmk or vehicle then exposed to ConA (125 nM), STS (250 nM), hydrogen peroxide (150 µM), A 1-42 (50 µM), or
A
25-35 (25 µM). Data shown are means ± S.E. *, p < 0.01 versus control; +,
p < 0.01 versus vehicle; 2-way ANOVA with
Tukey HSD post-hoc tests.
To differentiate pathways of apoptosis in PC12 cells, cells were transfected with an expression vector encoding bcl-2 or an empty vector control, and stable transfectants that expressed various levels of bcl-2 were selected (Fig. 3A). These cells lines were tested for their susceptibility to 2 prototypical apoptotic insults: STS and ConA. Cell lines that produced moderate levels of bcl-2, bcl-2#11 and bcl-2#12, were better protected from STS-induced apoptosis than from ConA-induced apoptosis (Fig. 3B). Cell lines expressing approximately 6-fold higher levels of bcl-2 (bcl-2#3 and bcl-2#10), however, were protected from apoptosis induced by both STS and ConA.
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An expression vector encoding crmA was used to transfect PC12 cells, and stable transfectants were selected and screened for their susceptibility to apoptotic insults. All of the crmA-transfected cell lines were protected from ConA-induced apoptosis (not shown). Two lines, crmA#3 and crmA#5, that showed the highest level of protection from ConA were used in subsequent assays. Neither crmA#3 nor crmA#5 cells were protected from STS-induced apoptosis (Fig. 4). Together, these results suggested that at least 2 pathways of apoptosis exist in PC12 cells. One pathway, activated by STS, is preferentially blocked by bcl-2 and is not blocked by crmA. The second pathway, activated by ConA, is blocked by crmA but is less efficiently blocked by bcl-2.
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To determine whether the apoptotic pathway activated by A is
sensitive to inhibition by bcl-2 or crmA, transfectants were exposed to
50 µM A
1-42. Cell viability was assessed
by nuclear morphology, because A
causes rapid decreases in the MTT assay that are not associated with cell death (Refs. 24 and 25, and
data not shown). Approximately 50% of control cells were apoptotic
after exposure to A
1-42. Significantly, crmA#3 and
crmA#5 cell lines were protected from this cell death, while bcl-2#11
and bcl-2#12 cell lines expressing moderate levels of bcl-2 were as
susceptible to A
1-42 as control cells (Fig.
5A). Bcl-2#3 and bcl-2#10 cell
lines expressing high levels of bcl-2 were also protected from
A
1-42 induced apoptosis. Because the
A
25-35 fragment of A
1-42 is toxic and has frequently been used in studies of the mechanisms of A
induced cell death (34), we wanted to determine whether crmA blocked cell death
induced by A
25-35. Surprisingly, although the bcl-2#3
and bcl-2#10 cell lines expressing high levels of bcl-2 were
protected from cell death induced by A
25-35, crmA transfectants were not protected from A
25-35 toxicity
(Fig. 5B).
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Cell death induced by A has been associated with oxidative damage to
cells (35-37), yet under at least some assay conditions antioxidants
protect against hydrogen peroxide- and iron-induced cell death, but do
not protect against A
-induced cell death (14). This suggests that
the mechanism of A
toxicity is less dependent on oxidative damage.
To determine whether bcl-2 and crmA transfectants were protected from
cell death induced by oxidative stress, cell survival was assessed
after exposure to hydrogen peroxide (Fig. 6A). As expected, cells
expressing bcl-2 were protected from cell death induced by this
oxidative insult, but crmA transfectants were not protected from cell
death. Moreover, in other experiments cell lines expressing moderate
levels of bcl-2 were observed to be protected from cell death induced
by the oxidative insults peroxynitrite and FeSO4, while
crmA transfectants were not protected from death. Cell viability after
exposure to 100 µM peroxynitrite was 95.4 ± 2.8%
in bcl-2 transfectants, 42.1 ± 1.5% in crmA transfectants, and
47.6 ± 0.8% in control transfectants (mean ± S.D.,
n = 2, p < 0.01 survival of bcl-2
transfectants versus control transfectants), while cell
viability after exposure to 120 µM FeSO4 was
94.5 ± 3.9% in bcl-2 transfectants, 46.3 ± 3.3% in crmA
transfectants, and 49.5 ± 4.8% in control transfectants
(mean ± S.D., n = 2, p < 0.01 survival of bcl-2 transfectants versus control
transfectants). These data show that bcl-2 levels sufficient to block
apoptotic pathway(s) activated by oxidative insults do not block the
apoptotic pathway activated by A
1-42, while crmA does
block the apoptotic pathway activated by A
1-42, but
does not block apoptotic pathway(s) activated by oxidative insults.
Therefore, these results strongly suggest that oxidative stress does
not mediate the initial activation of an apoptotic program by
A
1-42. Accordingly, antioxidants including GSH (Fig.
6B) and propyl gallate (data not shown) did not block cell
death induced by A
1-42 in these cells.
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Taken together, these results indicate that multiple apoptotic
pathways can be discerned in PC12 cells, and that the apoptotic pathway
activated by A1-42 is susceptible to inhibition by
crmA. However, although PC12 cells are often used to model aspects of
neuronal physiology, there is always the possibility that cell death
might be differentially regulated in immortalized "neuronal" cell
lines and neurons. Therefore, in order to confirm that crmA blocks the
apoptotic pathway activated by A
1-42 in neurons,
primary cultures of hippocampal neurons were transduced with crmA using
a herpes simplex virus amplicon containing an IRES-lacZ reporter.
Infected cells were identified by their expression of
-galactosidase
and their viability determined by nuclear morphology (Fig.
7). Cells infected with the control
IRES-lacZ amplicon and uninfected cells were similarly susceptible to
apoptosis induced by ConA, STS, A
1-42, and
A
25-35 (Fig. 8). However, as predicted, neurons transduced with crmA were protected from cell
death induced by ConA and A
1-42, but were not protected from cell death induced by STS or A
25-35 (Fig. 8). In
other experiments, hippocampal neurons transduced with bcl-2 were
protected from cell death induced by each of these insults (data not
shown). Because the IE 4/5 promoter used in the amplicon directs a high level of gene expression in neurons (38), these results appear consistent with the results obtained in PC12 cells expressing high
levels of bcl-2.
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DISCUSSION |
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The accumulation of plaques containing A is an invariant
feature of AD pathology, and there is abundant evidence suggesting that
A
contributes to the etiology of AD. Most significantly, chromosome
21-linked familial AD is caused by amyloid precursor protein alleles
with mutations in or near the A
coding sequence (39), and transgenic
mice expressing these amyloid precursor protein alleles exhibit
pathological changes and memory deficits reminiscent of AD (40, 41).
Furthermore, AD is characterized by a profound loss of neurons in
susceptible regions of the brain (1), and A
causes the apoptotic
death of cultured neurons (5, 7, 8). Therefore, understanding the
mechanisms by which A
induces apoptosis may be relevant to discovery
of clinical interventions to delay or alleviate AD.
We have previously suggested that cell death induced by A may
represent a type of APCD (10, 42). Neurons have been shown to be
susceptible to APCD: transforming growth factor
causes the
apoptotic death of cerebellar granule neurons (43), and ConA, a lectin
which binds mannose and cross-links glycoproteins on the cell surface,
causes the apoptotic death of cortical neurons (42). Other insults
including uv radiation (44) and growth factor withdrawal (45, 46) may
also cause apoptosis through APCD. However, apoptosis has also been
demonstrated in neurons exposed to oxidative stress (47), etoposide
(48), heat shock (49), and decreased extracellular K+ (50), indicating
that non-APCD apoptotic pathways also exist in neurons. Alternate
pathways of apoptosis can be discerned by their differential
sensitivity to blockade by bcl-2, crmA, and various caspase inhibitors
(20, 51). Anti-apoptotic members of the bcl-2 family of proteins are
very effective in blocking apoptosis induced by oxidative stress
(30, 52) and STS (26), while the viral serpin and caspase inhibitor
crmA effectively inhibits Fas- and TNF-mediated apoptosis (17). Both
bcl-2 and crmA, however, block apoptosis prior to activation of
caspase-3 (25, 53). Therefore, although multiple pathways are involved
in the induction of apoptosis, these pathways converge on the
activation of caspases which are the effectors of apoptosis.
The results presented here demonstrate that in PC12 cells, ConA and STS activate separate apoptotic pathways that are differentially blocked by crmA and bcl-2. Because the physiological target of crmA is likely to be caspase-8, the apical caspase activated by Fas and TNFR (18), the ability of crmA to inhibit ConA-induced apoptosis in PC12 cells suggests the possibility that ConA might cross-link death domain containing receptors of the Fas/TNFR family to initiate a death program. In addition to inhibiting Fas- and TNF-initiated apoptosis, crmA inhibits anoikis (54), apoptosis induced by NGF withdrawal from sympathetic neurons (55), and apoptosis induced by serum withdrawal from PC12 cells.2 These insults may also cause death through inappropriate activation of cell surface receptors. For example, death produced by NGF withdrawal has been proposed to be mediated by the low affinity NGF receptor (p75 NGFR), a member of the fas/TNF superfamily of receptors that has been reported to cause apoptosis in the absence of NGF (45).
Significantly, expression of crmA inhibited apoptosis induced by
A1-42. The ability of crmA to block
A
1-42-induced cell death supports the suggestion that
A
1-42 stimulates APCD by binding to and cross-linking
transmembrane receptor(s) (10). Interestingly, expression of crmA did
not inhibit apoptosis induced by A
25-35. Because
A
25-35 often appears more potent than
A
1-42 and causes more rapid cell death (56), it is
possible that levels of crmA were insufficient to block
A
25-35 toxicity. Alternatively, the mechanisms of A
25-35 toxicity in PC12 cells may differ from the
mechanisms of A
1-42 toxicity. Because previous studies
have also suggested the possibility that A
25-35 and
A
1-42 may have distinct mechanisms of toxicity (56,
57), caution should be used in interpreting the results of
investigations which use only the non-physiological
A
25-35 to study A
toxicity.
In a previous study, overexpression of bcl-2 did not inhibit
A25-35-induced death of PC12 cells or of human
neuroblastoma IMR-5 cells (58). Cell death was assessed by the MTT
assay, however, which has been shown to be an inaccurate indicator of cell death induced by A
(59, 60). The results shown here demonstrate
that the ability of bcl-2 to inhibit A
-induced death of PC12 cells
is dependent on its level of expression: relatively high levels of
bcl-2 expression are required to block apoptosis induced by
A
25-35 or A
1-42. Interestingly,
moderate levels of bcl-2 sufficient to inhibit apoptosis induced by
oxidative insults in PC12 cells were not able to inhibit apoptosis
induced by A
1-42. Moreover, and in agreement with the
results we have obtained in primary cultures of hippocampal neurons
(14), antioxidants did not block apoptosis induced by
A
1-42 or A
25-35. In contrast, a recent
study demonstrated that A
25-35 toxicity in PC12 cells
was blocked by antioxidants (37). As previously discussed (14),
discrepancies in the effectiveness of antioxidants in blocking A
toxicity may be related to methodological differences between
experiments. In the experiments described here, cells were exposed to
insults in Opti-MEM, a medium which supports the growth of the cells
for at least several days. These cells might be healthier and more
resistant to oxidative damage than cells assayed after serum and NGF
withdrawal into RPMI (37), since PC12 cells deprived of trophic support
in this manner undergo apoptosis within 2 to 3 days (61, 62).
There has been some ambiguity in the literature as to whether bcl-2 and related anti-apoptotic members of this family of proteins are able to block Fas- or TNF-induced apoptosis (19-24). The results here demonstrate that PC12 cells expressing moderate levels of bcl-2 were preferentially protected against apoptosis induced by oxidative insults and STS, but cells expressing higher levels of bcl-2 were also completely protected against apoptosis induced by ConA. These results suggest that the level of bcl-2 expression may be critical to its ability to suppress APCD, and that the ability of bcl-2 to block Fas- or TNF-induced apoptosis is likely to require high levels of expression.
It is not known whether apoptotic pathways in neurons are identical to
those demonstrated here in PC12 cells. However, because apoptotic
execution may involve inappropriate activity of proteins involved in
the regulation of the cell cycle (63, 64), it has been suggested that
differentiated and cycling cells use similar apoptotic programs, and
what varies is whether new protein synthesis is required or whether the
necessary proteins are available because they are constitutively made
in cycling cells (63). In fact, our results demonstrate that crmA
protects hippocampal neurons as well as PC12 cells from death induced
by ConA and A1-42, but does not block cell death
induced by STS or A
25-35 in either type of cell
culture. Therefore, our results suggest that central nervous system
neurons and PC12 cells use similar apoptotic pathways, and that
A
1-42 causes APCD in neurons as well as in PC12 cells.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant RO1-AG13007.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 949-824-6071; Fax: 949-824-2071; E-mail: kjivins{at}uci.edu.
The abbreviations used are:
AD, Alzheimer's
disease; A,
-amyloid; APCD, activation-induced programmed cell
death; STS, staurosporine; ConA, concanavalin A; IRES, internal
ribosome entry site; GSH, glutathione ethyl ester; DMEM, Dulbecco's
modified Eagle's medium; NGF, nerve growth factor; TNF, tumor necrosis
factor; MTT, 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide.
2 K. J. Ivins, unpublished observations.
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REFERENCES |
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