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The Journal of Neuroscience, February 15, 2000, 20(4):1386-1392
Caspase-2 Mediates Neuronal Cell Death Induced by  -Amyloid
Carol M.
Troy,
Sylvia A.
Rabacchi,
Wilma J.
Friedman,
Thierry F.
Frappier,
Kristy
Brown, and
Michael L.
Shelanski
Department of Pathology, Taub Institute for the Study of
Alzheimer's Disease and the Aging Brain and the Center for
Neurobiology and Behavior, Columbia University College of Physicians
and Surgeons, New York, New York 10032
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ABSTRACT |
 -amyloid (A) has been proposed to play a role in the
pathogenesis of Alzheimer's disease (AD). Deposits of insoluble A are found in the brains of patients with AD and are one of the pathological hallmarks of the disease. It has been proposed that A
induces death by oxidative stress, possibly through the generation of
peroxynitrite from superoxide and nitric oxide. In our current study,
treatment with nitric oxide generators protected against A-induced
death, whereas inhibition of nitric oxide synthase afforded no
protection, suggesting that formation of peroxynitrite is not critical
for A-mediated death. Previous studies have shown that aggregated
A can induce caspase-dependent apoptosis in cultured neurons. In all
of the neuronal populations studied here (hippocampal neurons,
sympathetic neurons, and PC12 cells), cell death was blocked by the
broad spectrum caspase inhibitor
N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone and more specifically by the downregulation of caspase-2 with antisense oligonucleotides. In contrast, downregulation of caspase-1 or caspase-3 did not block A1-42-induced
death. Neurons from caspase-2 null mice were totally resistant to
A1-42 toxicity, confirming the importance of this
caspase in A-induced death. The results indicate that caspase-2 is
necessary for A1-42-induced apoptosis in
vitro.
Key words:
-amyloid; neuronal cell death; caspases; caspase-2; hippocampal neurons; PC12 cells; sympathetic neurons
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INTRODUCTION |
The histopathological hallmarks of
Alzheimer's disease (AD) include the formation of neuritic plaques and
neurofibrillary tangles, and the loss of synapses (Masters et al.,
1985 ; Selkoe, 1990, 1997). Although the temporal order in which these
events occur and their relationship to one another is not clear, a
large body of evidence points to a toxic effect of -amyloid (A),
the major protein component of the senile plaque, on neurons (Yankner, 1996; Selkoe, 1997). In cell culture studies, a variety of effects of
-amyloid have been reported. These include induction of apoptotic neuronal death (Ii et al., 1996; Estus et al., 1997), as well as a
partial apoptotic program resulting in neuritic changes (Ferreira et
al., 1997; Mattson et al., 1998). A1-42 has
been proposed to cause death by regulation of components of the
apoptotic pathway (Estus et al., 1997), to induce oxidative stress
(Pike et al., 1997), and to cause death by free radical-mediated
pathways (Keller et al., 1998; Guo et al., 1999). None of these studies
has identified obligate mechanisms for A-induced apoptosis. Because
synaptic loss, neuritic changes, and cell loss are all features of
Alzheimer's disease, activation of the apoptotic cascade, especially
the activation of caspases, could explain many of the features of the
disease and its progression. Knowledge of which of the 14 known
mammalian caspases (for review, see Ahmad et al., 1998; Hu et al.,
1998; Humke et al., 1998; Thornberry and Lazebnik, 1998) are activated in response to A and which among these lead to neuritic alterations and apoptotic death will define specific pathways of cellular damage
and suggest potential targets for therapeutic intervention. The work
reported here examines which of the caspases is required for A to
induce apoptosis.
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MATERIALS AND METHODS |
Cell culture
PC12 cells. PC12 cells were grown as described
previously (Greene and Tischler, 1976; Troy et al., 1997) on rat tail
collagen-coated dishes in RPMI 1640 medium (Life Technologies,
Gaithersburg, MD) containing 5% fetal calf serum and 10%
heat-inactivated horse serum (complete medium). NGF-primed (neuronally
differentiated) PC12 cells were grown for at least 7 d in RPMI
1640 medium plus 1% horse serum and NGF (100 ng/ml). For cell survival
assays, cells (either naive or NGF-pretreated) were extensively washed in RPMI 1640 medium containing 1% fetal calf serum and replated on
fresh collagen-coated 24-well dishes in RPMI 1640 medium 1% FCS, with
NGF for primed cells. Various concentrations of
A1-42 were included in the medium as
indicated. Numbers of viable cells per culture were determined by
quantifying intact nuclei as described previously (Troy et al., 1997).
Counts were performed on triplicate cultures and reported as mean ± SEM.
Sympathetic neurons. Sympathetic neuron cultures were
prepared from 2-d-old rat pups as described previously (Troy et al., 1997) or from 2-d-old caspase-2  / and wild-type mouse pups
(Bergeron et al., 1998) (generous gifts from L. Bergeron and J. Yuan,
Harvard University, Boston, MA). Cultures were grown in 24-well
collagen-coated dishes in RPMI 1640 medium plus 10% horse serum with
mouse NGF (100 ng/ml). One day after plating, uridine and
5-fluorodeoxyuridine (10 µM each) were added to
the cultures and left for 3 d to eliminate non-neuronal cells
(<1% non-neuronal cells remain after 3 d). On the sixth day
after plating, A1-42 was added. Each culture was scored as described previously (Troy et al., 1997), as numbers of
living, phase-bright neurons counted in the same field at various times. Three replicate cultures were assessed for each condition, and
data are normalized to numbers of neurons present in each culture at
the time of A1-42 addition and reported as
mean ± SEM.
Hippocampal neurons. Primary cultures of dissociated
hippocampal neurons were prepared from embryonic day 18 (E18) rats
(Farinelli et al., 1998). E18 hippocampi were dissected, dissociated,
and maintained in a serum-free environment. Medium consists of a 1:1 mixture of Eagle's MEM and Ham's F12 supplemented with glucose (6 mg/ml), putrescine (60 µM), progesterone (20 nM), transferrin (100 µg/ml), selenium (30 nM), penicillin (0.5 U/ml), and streptomycin (0.5 µg/ml). Dissociates grown in this medium contain <2% glial cells
after 1 week. Cells were treated with A1-42
after 3-5 d in culture. Survival was quantified as described above for PC12 cells.
Preparation of amyloid
Lyophilized, HPLC-purified
-amyloid1-42 was purchased from D. Teplow
(Harvard University), and reverse A42-1 was
from Bachem (Torrance, CA). Peptides were reconstituted in sterile
water at a concentration of 400 µM. Aliquots of stocks were incubated at 37°C for 3 d to form aggregated amyloid.
Bioassay of NGF
Aliquots of RPMI with NGF (100 ng/ml) with and without
A1-42 (10 µM) were incubated at
37°C for 30 min and then spun down. The supernatant was added to PC12
cells deprived of trophic factors as described previously (Greene et
al., 1998). Cells were grown for 1 d, and survival was quantified
as described above.
Superoxide dismutase-specific activity
Cells were extracted with 0.5% NP-40, and protein was measured
by the Bradford method (Troy and Shelanski, 1994). Total and manganese-superoxide dismutase (Mn-SOD) levels were determined with a
modification of the xanthine-xanthine oxidase system, measuring the
reduction of nitroblue tetrazolium (NBT) at 560 nm in the absence and
presence of potassium cyanide (KCN) (Troy and Shelanski, 1994).
Briefly, cell extracts or SOD (Sigma, St. Louis, MO) were incubated in
50 mM sodium carbonate buffer at pH 10.2 containing 0.1 mM EDTA, 1 × 10 4
M xanthine, 1 mM KCN, 2.5 × 105 M NBT, and 2.2 × 109 M xanthine oxidase in a
volume of 1 ml. Reduction of NBT was measured at 560 nm. Total SOD
activity was determined from an SOD standard curve in the absence of
KCN. Copper/zinc-SOD is inhibited by KCN. Thus, only Mn-SOD activity
remains in the presence of KCN; Mn-SOD activity is reported as the
KCN-insensitive activity.
Caspase activity assay
Preparation of cell lysates. At 6 hr after
A1-42 treatment, cells were harvested for
assays of aspartase activity or Western blotting. Cells were rinsed in
cold PBS and then collected in a buffer of 25 mM
HEPES, pH 7.5, 5 mM EDTA, 1 mM EGTA, 5 mM MgCl2, 5 mM DTT, 10 µg/ml each of pepstatin and
leupeptin, and 1 mM PMSF. The cellular material
was left for 20 min on ice and then sonicated on ice. The lysate
was centrifuged for 20 min at 160,000 × g, and the
supernatant was frozen with liquid nitrogen and stored at 80°C
(Stefanis et al., 1996).
Cleavage of fluorogenic substrates. Lysates (25 µg
of protein) were incubated at 37°C in a buffer of 25 mM HEPES, pH 7.5, 10% sucrose, 0.1%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 10 mM DTT with the fluorogenic substrates
N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (DEVD-AFC) (15 µM) or
benzyloxycarbonyl-Tyr-Val-Ala-Asp-7-amino-4-trifluoromethylcoumarin (YVAD-AFC) (25 µM) (Enzyme Systems Products, Dublin, CA).
Cleavage of the substrates emitted a fluorescent signal that was
measured in a Perkin-Elmer (Emeryville, CA) LS-50B fluorometer
(excitation of 400 nm, emission of 505 nm).
Synthesis of antisense oligonucleotides
Oligonucleotides bearing an SH group at their 5' end and an NH
group at their 3' end were purchased from Operon (Alameda, CA). As
described previously (Troy et al., 1996a), oligonucleotides were
resuspended in deionized water, an equimolar ratio of Penetratin 1 (Oncor Inc., Gaithersburg, MD) was added, and the mixture was incubated
at 37°C for 1 hr. The yield of the reaction, estimated by SDS-PAGE
followed by Coomassie blue staining, was routinely above 50%. As a
control, a scrambled sequence of the antisense oligonucleotide (same
base composition, different order) was used. Antisense sequences used
were as follows: ACasp1, CCTCAGGACCTTGTCGGCCAT; ACasp2,
GCTCGGCGCCGCCATTTCCCAG; and ACasp3, GTTGTTGTCCA TGGTCACTTT.
Western blotting
Neuronal cells were harvested in lysis buffer as described above
or in SDS-containing sample buffer and immediately boiled. Equal
amounts of protein were separated by 15% PAGE, transferred to
nitrocellulose, and immunostained as described previously (Stefanis et
al., 1997). Anti-caspase-1 (Transduction Laboratories, Lexington, KY)
was used at a dilution of 1:500. Anti-caspase-2 (Troy et al., 1997) was
used at a dilution of 1:250. Anti-caspase-3 was a generous gift from
J. L. Goldstein (University of Texas Southwestern Medical Center,
Dallas, TX) (Wang et al., 1996) and was used at a dilution of
1:1000. Visualization was with ECL using goat-anti-rabbit peroxidase at
1:1000. The relative intensity of the protein bands was quantified using Scion NIH Image 1.55 software.
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RESULTS |
-Amyloid-induced neuronal cell death is not mediated
by peroxynitrite
We studied A-induced death in three different neuronal cell
types: PC12 cells, the most widely used neuronal cell line; sympathetic neurons, the neuron for which PC12 cells are a model; and hippocampal neurons, because the hippocampus is affected extensively in AD. Previous studies reporting neurotoxicity of A have used a wide range
of concentrations of different forms of A (25-35, 1-40, and 1-42)
in a variety of cell types, including cortical neurons, hippocampal
neurons, and cultured cell lines (Pike et al., 1991a,b; Ii et al.,
1996; Estus et al., 1997; Jordan et al., 1997; Kruman et al., 1997). We
have performed dose-response studies with aggregated A1-42 in PC12 cells, sympathetic neurons, and
hippocampal neurons (Fig.
1A) to determine
whether these three neuronal cell types respond in a similar manner. At
a concentration of 10 µM, there was equivalent
survival (~50%) of all three neuronal cell types after 1 d of
exposure (Fig. 1A). No death was seen in PC12 cell
cultures treated with the same concentrations of
A42-1, the inactive reverse sequence of A
(data not shown), supporting a specific effect of
A1-42. All subsequent experiments were done
with 10 µM A1-42.
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Figure 1.
Nitric oxide protects against -amyloid-induced
death in neuronal cells. A, A1-42
induces dose-dependent death in three different neuronal cell types.
E18 hippocampal neurons were grown in culture for 3 d and then
exposed to increasing concentrations of A1-42. Survival
was assessed after 1 d by counting nuclei in cell lysates
(n = 3). Survival is reported relative to untreated
cultures and is given as mean ± SEM. Sympathetic neurons were
grown in culture for 5 d and then exposed to increasing
concentrations of A1-42. Survival was assessed after
1 d by counting cells in the living cultures. Survival is reported
relative to that in the same cultures before A1-42
treatment and is given as mean ± SEM (n = 3).
PC12 cells were exposed to increasing concentrations of
A1-42. Survival was assessed after 1 d by counting
nuclei in cell lysates (n = 3). Survival is
reported relative to untreated cultures and is given as mean ± SEM. These are representative experiments. Comparable results were
obtained in six additional independent experiments with hippocampal
neurons, in three additional experiments with sympathetic neurons, and
in five additional experiments with PC12 cells. B,
Mn-SOD is not induced by A1-42 treatment. PC12 cells
were treated with or without A1-42 (10 µM) for 6 hr (n = 3). Cells were
extracted with 0.5% NP-40, and protein
was measured by the Bradford method. Total SOD and
Mn-SOD levels were determined by the xanthine-xanthine oxidase system,
with measurement of the reduction of nitroblue tetrazolium at 560 nm in
the presence and absence of KCN. Mn-SOD activity was determined from an
SOD standard curve and is reported as the KCN-insensitive activity ± SEM. C, Increasing NO protects from
A1-42-induced neuronal cell death. PC12 cells and
sympathetic neurons were exposed to A1-42 (10 µM) in the presence or absence of SNAP (100 µM) or L-NAME (10 µM).
Survival was assessed after 1 d as described above
(n = 3). This is a representative experiment;
comparable results were obtained in three additional independent
experiments. Survival is reported relative to untreated cultures and is
given as mean ± SEM. Similar results were obtained with cultured
sympathetic neurons.
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It has been proposed that A toxicity is caused by the
induction of oxidative stress (Ii et al., 1996; Kruman et al., 1997; Pike et al., 1997; Keller et al., 1998; Guo et al., 1999).
Specifically, A-treated PC12 cells have been shown to produce
peroxynitrite (ONOO), a toxic product of
the superoxide anion
(O2), and nitric oxide
(NO), and to be protected from A toxicity by the overexpression of
Mn-SOD the inducible, manganese-dependent form of superoxide dismutase
normally expressed in the mitochondria (Keller et al., 1998). Mn-SOD
has been shown to be increased in response to an increase in
O2 (Troy and Shelanski,
1994). Thus, changes in the specific activity of Mn-SOD may afford an
indication of O2 levels
in the cells. Treatment with A1-42 for 6 hr
had no effect on total SOD or Mn-SOD activities in our cultures (Fig. 1B). The other component of
ONOO, NO, has been shown to be both
neurotoxic and neuroprotective depending on the type of insult to which
the cell has been exposed. If peroxynitrite is a component of the A
death pathway, then inhibition of NO production should be protective.
We have examined the role of NO in
A1-42-treated PC12 cells and sympathetic neurons by inhibiting the generation of endogenous NO with
N-nitro-L-arginine methyl ester
(L-NAME) (10 µM), a
general inhibitor of nitric oxide synthase, and by treating the cells
with the NO generator S-nitroso penicillamine (SNAP)
(100 µM) (Fig. 1C). The
concentrations of L-NAME and SNAP were selected
based on previous work by us and by our colleagues using PC12 cells and
sympathetic neurons (Farinelli et al., 1996; Troy et al., 1996a).
Inhibition of endogenous NO generation by L-NAME
did not protect PC12 cells or sympathetic neurons in the presence of
A1-42. Conversely, concurrent treatment with
the exogenous NO generator SNAP led to complete protection in these
neurons. SNAP alone was toxic to hippocampal neurons. This protective
effect of NO is also seen in PC12 cells and sympathetic neurons
deprived of NGF (Farinelli et al., 1996), a death paradigm that has
been shown to require cell cycle elements.
The similarity between the protection profiles in
A1-42 exposure and trophic factor deprivation
led us to examine whether agents that block cell cycle progression also
protect from A1-42. Hippocampal neurons and
PC12 cells were treated with A1-42 in the
presence or absence of the cell cycle inhibitor flavopiridol. Flavopiridol is a flavonoid derivative that inhibits cyclin-dependent kinase 1 (cdk1), cdk2, and cdk4 activities (Losiewicz et al., 1994; De Azevedo et al., 1996) and is reported to block progression from G1 to S and G2 to M phases of the cell cycle (Kaur et al., 1992;
Vesely et al., 1994). Flavopiridol provided protection against A1-42 for both hippocampal neurons and PC12
cells (Fig. 2A). This
is in accord with the recent data that elements of the cell cycle are
required for 30 µM
A1-40 to induce death in cortical neurons
(Giovanni et al., 1999).
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Figure 2.
A, The cell cycle inhibitor
flavopiridol protects hippocampal neurons and neuronal PC12 cells from
A1-42 toxicity. Hippocampal cultures and neuronal PC12
cells were treated with A1-42 in the presence or
absence of flavopiridol (1 µM) (n = 3). Survival was assessed after 1 d as described in Figure 1, is
reported relative to untreated cultures, and is given as mean ± SEM. This is a representative experiment; comparable results were
obtained in six additional independent experiments for hippocampal
cultures and three additional experiments for PC12 cells.
B, A1-42 does not inhibit NGF activity.
RPMI with NGF was incubated with or without A1-42 (10 µM) for 30 min at 37°C, and A1-42 was
removed by centrifugation. The various media were added to PC12 cells,
which had been subjected to trophic factor deprivation. Survival was
quantified at 1 d and is given as mean ± SEM
(n = 3).
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The above lines of evidence support a similar mechanism of death for
A and trophic factor deprivation. Because
A1-42 induces cell death in primed PC12 cells
and sympathetic neurons in the presence of NGF, we considered the
possibility that A might bind to NGF in the media and effectively
inactivate it, resulting in trophic factor withdrawal. Using an
established bioassay for NGF (Greene et al., 1998), we determined that
the neurotrophic activity of NGF was not diminished by preincubation
with A1-42 in the media (Fig.
2B).
A1-42-induced cell death requires caspase-2
Death induced by A is inhibited by the broad spectrum caspase
inhibitor N-benzyloxycarbonyl-val-ala-asp-fluoromethyl
ketone (zVAD-FMK), demonstrating that caspase activity is
essential for A-induced apoptosis (Jordan et al., 1997; Guo et al.,
1999). Treatment of hippocampal neurons or PC12 cells with
A1-42 induced caspase activity within 6 hr,
as detected by cleavage of DEVD-AFC (Fig.
3A), a substrate for caspases
related to caspase-3. This peptide is not cleaved by caspase-2 and
minimally cleaved by caspase-1 family members (Talanian et al., 1997;
Thornberry et al., 1997). The DEVD-AFC cleavage activity was completely
prevented by simultaneous treatment of the cultures with A and 10 µM DEVD-FMK, the pseudosubstrate inhibitor
(Fig. 3A). There was no cleavage of YVAD-AFC, a substrate
for caspase-1 family members, by the same cell lysates (data not
shown). No specific substrate is available for caspase-2. Differential
use of caspase inhibitors can provide some information about caspase
requirements for a particular mode of death. In the studies reported
here, we have used several different competitive irreversible
pseudosubstrate caspase inhibitors: YVAD-FMK, which inhibits caspase-1,
-4, and -5; and DEVD-FMK, which is moderately specific for members of
the caspase-3 family [including caspase-3 and -7 (Talanian et al.,
1997; Thornberry et al., 1997)] when used at low concentrations (10 µM) and the broad spectrum inhibitor zVAD-FMK.
Surprisingly, DEVD-FMK provided no protection against A-induced
neuronal cell death (Fig. 3B), despite the complete prevention of the DEVD cleaving activity by this concentration of the
inhibitor (Fig. 3A). No rescue from A-induced death was seen with YVAD-FMK (100 µM) (Fig.
3B) in any of the three neuronal types. Thus, it is unlikely
that caspase-1, -3, or -7 are required for the A apoptotic pathway
(caspase-4 and -5 have not been found in rodent cells; J. Angelastro,
personal communication). On the other hand, zVAD-FMK gave complete
protection (Fig. 3B), confirming that A induces a
caspase-mediated death.
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Figure 3.
A, A1-42 induces
caspase activity in hippocampal neurons and PC12 cells. Hippocampal
neurons and neuronal PC12 cells were treated with A1-42
(10 µM) with or without DEVD-FMK (10 µM)
for 6 hr. Cells were lysed, and 25 µg of protein of each treatment
was incubated with the fluorogenic substrate DEVD-AFC (15 µM). The release of AFC was quantified in an LS50B
fluorometer. This is a representative experiment; comparable results
were obtained in three additional independent experiments.
B, Differential protection by caspase inhibitors from
A1-42-induced death. Cultures of hippocampal neurons,
PC12 cells, and sympathetic neurons were exposed to
A1-42 (10 µM) in the presence or absence
of the indicated inhibitors (n = 3): YVAD-FMK at
100 µM, DEVD-FMK at 10 µM, and zVAD-FMK at
50 µM. Cells were counted after 1 d as described in
Figure 1. Survival is reported relative to untreated cultures and is
given as mean ± SEM.
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Because the inhibitors can only provide circumstantial evidence about
specific caspase activation during death, we used Western blotting with
antibodies specific for caspase-1, -2, and -3 to determine whether any
of these caspases was activated by A1-42 treatment. All three of the caspases were detected in untreated hippocampal neurons (Fig. 4), as well as
in PC12 cells and sympathetic neurons (Troy et al., 1997) (data not
shown). Hippocampal neurons were treated with
A1-42 for 2, 6, and 18 hr, and cell lysates were analyzed by Western blotting. The antiserum to caspase-2 detects
the proform and a p37 peptide, which is an intermediate cleavage
product in the formation of the p20 active peptide (Stefanis et al.,
1997, 1998). The p37 peptide was detected after 6 hr treatment, a time
at which there was a concomitant decrease in the proform caspase-2p51
(Fig. 4A). Caspase-3 also showed an increase in
appearance of the p18 active peptide (Fig. 4B),
consistent with the changes in DEVD-AFC cleaving activity described
above (Fig. 3A). However, the proform of caspase-3,
caspase-3p32, also increased over the time course (Fig.
4B). There was no activation of caspase-1 apparent at
6 hr (Fig. 4C), concurring with the lack of YVAD-AFC
cleaving activity in the lysates prepared at this same time.
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Figure 4.
Caspase-2 and caspase-3 are activated in
hippocampal neurons after A1-42 treatment. Hippocampal
cultures were incubated with or without A1-42 for the
indicated times. Cell lysates (equal amounts of protein, determined by
the Bradford method) were subjected to Western blotting using the
indicated antisera. Ponceau staining confirmed equal loading. These are
representative blots; comparable results were obtained in three
independent experiments. A, Caspase-2. B,
Caspase-3. C, Caspase-1.
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To assess the role of each of these caspases in mediating
A1-42-induced death Penetratin1-conjugated
antisense oligonucleotides were used to downregulate caspase-1,
caspase-2, and caspase-3 independently. Each oligonucleotide
specifically downregulates the respective caspase by at least 70% in
PC12 cells after 6 hr treatment (Fig.
5A). There is no cross
regulation of the other caspases; that is, V-ACasp2 does not affect
caspase-1 or caspase-3 levels, etc. (Troy et al., 1997) (data not
shown). Scrambled oligonucleotides (same base composition) had no
effect on protein levels (Troy et al., 1997) (data not shown). When the
three antisense oligonucleotides were tested, only the antisense
caspase-2 (V-ACasp2) (Fig. 5B) protected from
A1-42 damage; cells were treated
simultaneously with the antisense oligonucleotides and
A1-42. The requirement for caspase-2 in
A-induced death was confirmed using cultured sympathetic neurons
from caspase-2 null mice (Fig. 6).
Sympathetic neurons from postnatal day 1 wild-type and caspase-2
null mice (Bergeron et al., 1998) were grown in culture for 5 d
and then treated with A1-42 (10 µM), and survival was quantified daily. Neurons
from wild-type mice had 55% survival after 1 d and only 25%
survival after 4 d treatment. Neurons from caspase-2 null mice
were completely resistant to A1-42 treatment, even after 4 d of exposure (Fig. 6). The sympathetic neurons from caspase-2 null mice were also resistant to 30 µM A1-42, a
concentration that gave 20% survival of wild-type neurons after 1 d of treatment, and no survival after 4 d treatment (data not shown).
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Figure 5.
Caspase-2 is necessary for
A1-42-induced neuronal cell death. A,
Specific downregulation of caspase-1, -2, or -3. PC12 cells were
treated with the indicated antisense oligonucleotides (240 nM) for 6 hr. Cells lysates were subjected to Western
blotting using the appropriate antisera, i.e., anti-caspase-1 for
V-ACasp1-treated cells. B, Only downregulation of
caspase-2 protects against A1-42-induced neuronal cell
death. Cultures of hippocampal neurons, PC12 cells, and sympathetic
neurons were treated with 10 µM A1-42 in
the presence or absence of the indicated antisense oligonucleotides,
each at a concentration of 240 nM (n = 3). Survival was quantified after 1 d, is reported relative to
untreated cultures, and is given as mean ± SEM. This is a
representative experiment; comparable results were obtained in three
additional independent experiments with hippocampal cultures, as well
as three additional experiments each with PC12 cells and sympathetic
neurons.
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Figure 6.
Sympathetic neurons from caspase-2 null mice are
resistant to A1-42 toxicity. Sympathetic neuron
cultures from 1-d-old wild-type or caspase-2 null pups were treated
with A1-42 (n = 3). Survival was
quantified daily, as described in Figure 1. Survival is reported
relative to that in the same cultures before A1-42
treatment and is given as mean ± SEM (n = 3).
This is a representative experiment; comparable results were obtained
in four additional independent experiments.
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DISCUSSION |
The role of A in the pathogenesis and progression of
Alzheimer's disease has not yet been fully determined. It is clear
that deposits of insoluble A are found in plaques in the brains,
particularly the hippocampus, of patients with AD and that insoluble
A can induce apoptotic neuronal cell death in vitro
(Selkoe, 1990; Pike et al., 1991b). If indeed A plays an important
role in AD, knowledge of the mechanisms used by A to induce neuronal
cell death will identify potential molecular targets for development of
therapies for AD. Study of model systems of A-induced neuronal cell
death allows the delineation of the molecular pathways traversed by A to induce neuronal cell death. A variety of laboratories have presented work showing A induction of apoptosis in multiple cell types in culture (Pike et al., 1991a; Li et al., 1996; Estus et al.,
1997; Jordan et al., 1997; Pike et al., 1997; Mattson et al., 1998),
and apoptosis is seen in human AD brains as well (Cotman et al., 1994;
Cotman and Su, 1996). Recent work from our laboratory has shown that
differing insults to neurons result in activation of apoptotic
pathways, which use different caspases (Troy et al., 1996b, 1997) (Fig.
7). The studies presented here show that
A1-42 mediated death in three different
neuronal cell types requires the presence of caspase-2 and is
accompanied by caspase-2 activation. Although caspase-3 activation
occurs, it does not mediate cell death in this paradigm. The activation
of caspase-3 may be occurring in parallel with that of caspase-2, or
caspase-2 may be activating caspase-3. However, it is clear that the
activated caspase-3 is not executing death in our model. Caspases have
been classified in several ways, based on both structure and function.
Caspase-2 has been classified as either an effector, together with
caspase-3 and caspase-7 (Thornberry et al., 1997), or as an initiator
(Thornberry and Lazebnik, 1998). Our data would support a role for
caspase-2 as an initiator, which then activates other effector caspases or autoactivates so that caspase-2 can act as both initiator and effector to lead to death (Fig. 7).
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Figure 7.
Caspase specificities in different paradigms of
cell death. Schematic illustration of the pathways to cell death for
-amyloid, trophic factor deprivation, and free radical-mediated
oxidative stress.
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AD hippocampus has been shown to have both an increase in caspase-3
immunoreactivity (Masliah et al., 1998; Gervais et al., 1999), as well
as appearance of activated caspase-3 reactivity (Chan et al., 1999).
The work of Gervais (1999) showed that caspase-3 can cleave the
amyloid precursor protein and cause an increase in secretion of A,
measured as picomolar quantities in cell media; cell death was
not measured in that study. In our system, exogenous aggregated
A1-42 is added at a concentration of 10 µM, many fold higher than that produced by caspase-3
activation. Additionally, the A produced by caspase-3 would most
likely be in the less toxic soluble form over the time course of our
experiments. Thus, blockade of caspase-3 activity would be expected to
have little effect on cell survival in our model. In more chronic
paradigms, caspase-3 may play a larger role in potentiating death by
enhancing production of A. Additionally, caspase-3 activation could
play a role in proteolytic remodeling of the cytoskeleton and neuritic breakdown seen in these cells.
We found little evidence in our studies that free radicals play a key
role in A1-42-induced apoptosis in culture.
The lack of protection by the nitric oxide synthase inhibitor
L-NAME and the protection from death by SNAP argue against
peroxynitrite mediation of apoptosis. The protection by SNAP supports
the possibility that there is inhibition of caspase activity by
nitrosylation, as has been seen in other models of cell death (Mannick
et al., 1999). These findings do not preclude a contributory role for oxidative damage in Alzheimer's disease but argue against their role
in these acute apoptotic models.
The protection by SNAP, the NO generator, from
A1-42 toxicity and the requirement for
caspase-2 activity in this death pathway are elements shared with the
death pathway for trophic factor deprivation, a pathway that also uses
elements of the cell cycle. We have found that both hippocampal neurons
and neuronal PC12 cells were protected by flavopiridol, a cell cycle
inhibitor. This extends the recently published work showing protection
of cortical neurons from A1-40 death by
inhibition of the cell cycle (Giovanni et al., 1999).
The caspase specificities for different cell death paradigms are
presented schematically in Figure 7. By studying three paradigms in
different neuronal cells, we can conclude that caspase specificity is
determined by the death stimulus as opposed to the neuronal cell type.
Although there are similarities between death induced by
A1-42 and by trophic factor deprivation,
including protection by nitric oxide and by the cell cycle inhibitor
flavopiridol and use of caspase-2 as a mediator of cell death, there
are also differences. Most notable is the lack of protection against
A-induced death by NGF in PC12 cells and sympathetic neurons, as
well as the susceptibility of sympathetic neurons from caspase-2 null
mice to trophic factor deprivation (Bergeron et al., 1998).
Therefore, the death pathways for these two stimuli are not identical.
Our data using caspase inhibitors, specific antisense
oligonucleotides, and caspase-2 null mice implicate caspase-2 as a
mediator of A1-42-induced death. AD is both a
devastating disease and an increasing health problem. The development
of specific therapies that target caspase-2 may allow more effective
treatment for AD.
| |
FOOTNOTES |
Received Oct. 11, 1999; revised Nov. 30, 1999; accepted Dec. 7, 1999.
This work was supported by grants from the National Institutes of
Health (C.M.T., W.J.F., M.L.S.) and the Muscular Dystrophy Association
(C.M.T.). We thank Christine Le for technical assistance.
Correspondence should be addressed to Carol M. Troy, College of
Physicians and Surgeons of Columbia University, Department of
Pathology, 630 West 168th Street, New York, NY 10032. E-mail: cmt2{at}columbia.edu.
| |
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