Received for publication, November 26, 2002, and in revised form, January 28, 2003
Many cases of autosomal dominant early onset
familial Alzheimer's disease result from mutations in
presenilin-1 (PS1). In this study, we examined the role of the
PS1 homologue gene sel-12 of Caenorhabditis
elegans under oxidative stress and clarified the
sel-12-induced apoptosis. A genetic null allele mutant,
sel-12(ar171), showed resistance to oxidative
stress and prevented mitochondrial dysfunction-induced apoptosis. On
the other hand, another allele mutant,
sel-12(ar131), that carries a missense mutation
showed a proapoptotic activity, which may be the result of a gain of function property. Also, sel-12(ar131)-induced
apoptosis was ced-3- and
ced-4-dependent. Dantrolene, which specifically
inhibits Ca2+ release from endoplasmic reticulum stores,
prevents sel-12(ar131)-induced apoptosis.
SEL-12, which is localized in the endoplasmic reticulum, may induce
apoptosis through abnormal calcium release from the endoplasmic
reticulum. Together, with the previous finding that human PS1 could
substitute for SEL-12, these results suggest the similar involvement of
PS1-inducing apoptosis under oxidative stress and mitochondrial
dysfunction in the Alzheimer's Disease brain.
 |
INTRODUCTION |
Many cases of autosomal dominant early onset familial Alzheimer's
disease (FAD)1 result from
mutations in the gene encoding presenilin-1 (PS1) (1). PS1 is an
integral membrane protein comprising multiple transmembrane domains
that is expressed in neurons throughout the brain and primarily
localized in the endoplasmic reticulum (ER) and the Golgi apparatus (2,
3). PS1 is essential for
-secretase cleavage of the amyloid
precursor protein (APP) and Notch protein (4-7). FAD-related mutations
in PS1 increase the levels of A
42 in Alzheimer's disease brains
(8), transfected cell lines, and transgenic animals (9, 10). It is
recognized that oxidative stress and mitochondrial abnormalities may
play important roles in the pathogenesis of Alzheimer's disease (11, 12). Overexpression of PS1 mutations in cultured cells increases their
vulnerability to oxidative stress and A
toxicity (13, 14). As it is
well known that oxidative stress plays the role of apoptosis (15), the
oxidative stress and apoptosis may be also tightly linked in
Alzheimer's disease. In fact, FAD-related mutant PS1 is also involved
in apoptosis (13, 16).
Caenorhabditis elegans has the PS1 homologue gene
sel-12, which facilitates lin-12/Notch
receptor-mediated signaling, and the mutation of sel-12
produces defects in vulva and neuronal development (17, 18). Levitan
et al. (19) have shown that human PS1 could substitute for
C. elegans sel-12 protein. In this study, we try to
elucidate the possibility that sel-12 is involved in
oxidative stress and apoptosis in C. elegans. In attempting to clarify the relationship between sel-12 and cell death,
we used the C. elegans sel-12 and mev-1 mutants
in this study. The mev-1(kn1) mutant has a defect
in the cytochrome b large subunit in complex II of the
mitochondrial electron transport chain that shows hypersensitivity to
oxygen and paraquat (20). Senoo-Matsuda et al. (21) have
reported that the generation of reactive oxygen species is elevated in
mev-1 mutants. Furthermore, increased numbers of apoptosis
were observed in mev-1(kn1)
embryos.2 For these reasons,
we chose to investigate sel-12mev-1 double mutants in this
study. The sel-12 mutants examined in this study were
sel-12(ar131)unc-1(e538)
and
sel-12(ar171)unc-1(e538).
ar131 carries a missense mutation (C60S), and
ar171 carries a nonsense mutation (W225stop), which produces
a truncated SEL-12 protein. Genetically,
sel-12(ar171) represents a null allele (17). The marker mutant unc-1(e538) was also examined as a
control. Moreover, we demonstrate in this study that intracellular
calcium regulation is an important factor for sel-12-induced
apoptosis by the use of dantrolene to block calcium release from the
ER.
 |
EXPERIMENTAL PROCEDURES |
Nematode Propagation--
The C. elegans alleles used
were as follows: Bristol N2 wild-type strain,
unc-1(e538);
sel-12(ar131)unc-1(e538);
sel-12(ar171)unc-1(e538); ced-3(n1286);
ced-3(n1286)sel-12(ar131)unc-1(e538);
ced-4(n1894); ced-4(n1894)sel-12(ar131)unc-1(e538);
mev-1(kn1);
mev-1(kn1)lon-1(e185); and
sel-12(ar171)unc-1(e538)mev-1(kn1)lon-1(e185).
The N2 wild-type strain and unc-1(e538),
ced-3(n1286), and
ced-4(n1894) mutant strains used in this work
were obtained from the Caenorhabditis Genetics Center (CGC, St. Paul,
MN), which is funded by the National Institutes of Health, National
Center for Research Resources (NCRR, Bethesda, MD).
General standard methods were used for the strain maintenance and
culturing of C. elegans (22). In particular, animals were grown at 20 °C on NGM agar plates seeded with live bacteria
(Escherichia coli strain OP50) as food source.
Statistics--
Statistical analysis was done by unpaired
Student's t test with the exception of a comparison
analysis of apoptotic cells in sel-12 mutants, which was
done by one-way factorial analysis of variance.
Sensitivity of sel-12 Mutants to Paraquat--
Age-synchronous
hermaphrodites after maturation were prepared by the alkaline
hypochlorite method (23). 2-day-old synchronized animals were placed on
NG agar plates with OP50 and 40 µM 5-fluorodeoxyuridine, which was used to block progeny development (24). Seven days after
hatching, normally developed animals were transferred to NG agar plates
containing 5 mM paraquat in addition to
5-fluorodeoxyuridine and OP50. Animals were grown at 20 °C and
subsequently followed for survival. On the fifth day after exposure to
paraquat, the number of surviving animals was counted. The survival
rate of 60 animals of each strain was followed for each of the four
independent trials.
Sensitivity of sel-12 Mutants to Hydrogen
Peroxide--
Age-synchronous animals were grown in liquid culture (S
medium, 0.001% cholesterol, and Escherichia coli strain
NA22). Two days after eggs hatched, hydrogen peroxide was added at
several concentrations. After a 2-h exposure, the numbers of animals
surviving was counted. The survival rate of 100 animals of each strain
was followed for each of the four independent trials.
Cell Death Assays--
Embryonic development was followed
microscopically by using Nomarski optics, and apoptotic cells in the
comma embryonic stage were counted. Embryos were collected by the
alkaline hypochlorite method. The number of cell corpses was counted in
70 embryos for each of the three independent trials.
Dantrolene Treatment--
Synchronized animals were grown on NG
agar plates with 10 mM dantrolene. On the third day after
hatching, embryos were collected by the alkaline hypochlorite method.
Cell death in embryos was examined as described above.
 |
RESULTS |
sel-12(ar171) Mutants Show Resistance to Oxidative
Stress--
To investigate the relationship between oxidative stress
and sel-12, we examined the survival rate of
sel-12 mutants under oxidative stress. The toxicity of
paraquat and hydrogen peroxide is exerted by their capacity to generate
superoxide anions and hydroxy radicals (25, 26), which expose animals
to oxidative stress. We treated animals with 5-fluorodeoxyuridine to
prevent the nematodes from dying from bags of worms (the
sel-12 mutants have Egl phenotype and die from hatches of
eggs in their bodies without using 5-fluorodeoxyuridine). The survival
rate is given in Fig. 1,
A and B. Without paraquat,
sel-12(ar171)unc-1(e538) showed a similar survival curve when compared with the control strain
unc-1(e538) (Fig. 1A). In contrast,
sel-12(ar171)unc-1(e538) showed an increased survival rate in the presence of 5 mM
paraquat, and its survival rate at the fifth day after exposure to
paraquat was significantly elevated compared with that of
unc-1(e538) (Fig. 1, B and
C), whereas the survival rate of
sel-12(ar131) showed no significant change from
that of unc-1(e538) (Fig. 1, A-C). Treatment with 10 mM paraquat also caused
sel-12(ar171) to show a tendency for higher
resistance, although the difference was not statistically significant
(data not shown). Similarly,
sel-12(ar171)unc-1(e538) also showed elevated resistance to hydrogen peroxide compared with that
of unc-1(e538) (Fig. 1D).

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Fig. 1.
Survival curve of sel-12
mutants and unc-1(e538) adult
animals. 7-day-old synchronized animals were placed on NG agar
plates with 5 mM paraquat and subsequently followed for
subsequent survival. 5-Fluorodeoxyuridine was used to prevent the
development of progeny. A, survival curve under normal
condition. sel-12 mutants showed a similar survival rate
compared with unc-1(e538). B, survival
curve with 5 mM paraquat. The double mutant
sel-12(ar171)unc-1(e538)
showed an increased survival rate in the presence of paraquat compared
with unc-1(e538), whereas the survival rate of
sel-12(ar131) showed no significant change from
that of unc-1(e538). C, survival rate
on the fifth day after exposure to paraquat (12th day after hatching).
Error bar indicates ± S.D. Percent survival was
expressed as a relative value to the survival rate on the fifth day
under normal conditions. The increased survival rate of
sel-12(ar171)unc-1(e538)
compared with unc-1(e538) is statistically
significant. *, p < 0.01 versus
unc-1(e538). D, survival rate of
sel-12(ar171)unc-1(e538)
and unc-1(e538) adult animals in the presence of
hydrogen peroxide. Animals at 2 days of age were cultured in each concentration of hydrogen peroxide for 2 h.
Survival rate was immediately estimated after treatment. Percent
survival was expressed as a relative value to the survival rate without
hydrogen peroxide. The survival rate of
sel-12(ar171)unc-1(e538) is
elevated with any concentration of hydrogen peroxide compared with that
of unc-1(e538), and it was significantly elevated
in the presence of 300 mM hydrogen peroxide. *,
p < 0.01 versus
unc-1(e538).
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|
sel-12(ar171) Prevents mev-1(kn1)-induced Apoptosis--
We
constructed sel-12mev-1(kn1) double mutants to
clarify the role of sel-12 in inducing apoptosis under
endogenous oxidative stress and mitochondrial dysfunction. The number
of apoptosis in embryos of C. elegans is easily identified
with Nomarski optics (27, 28). The nucleus of the apoptotic cell
becomes refractile resembling a flat button (Fig.
2A). The increased numbers of
apoptosis were observed in mev-1(kn1) embryos
(Fig. 2B). The number of apoptosis in
sel-12(ar171)mev-1(kn1)
embryos was remarkably decreased, which means that
sel-12(ar171)
prevents-mev-1-induced apoptosis (Fig. 2B).

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Fig. 2.
A, representative Nomarski images
of apoptosis phenotype are shown for each strain. Cell corpses are
indicated by arrowheads. a,
mev-1(kn1)lon-1(e185);
b,
sel-12(ar171)unc-1(e538)mev-1(kn1)lon-1(e185),
and c,
sel-12(ar171)unc-1(e538).
B, the number of apoptosis in N2,
sel-12(ar171), mev-1(kn1),
and
sel-12(ar171)mev-1(kn1).
Embryos in the comma stage were scored for apoptotic cell corpses. The
number of apoptosis in mev-1(kn1) was increased
compared with wild-type N2. The number of apoptosis in the
sel-12(ar171)mev-1(kn1)
double mutant was decreased compared with that in
mev-1(kn1). *, p < 0.01 versus mev-1(kn1). Complete genotypes
are as follows:
sel-12(ar171)unc-1(e538) #;
mev-1(kn1)lon-1(e185) ##;
and sel-12(ar171)
unc-1(e538)mev-1(kn1)lon-1(e185)
###.
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|
The Number of Apoptosis in sel-12(ar131) Embryos Is Significantly
Elevated--
The number of apoptosis in
sel-12(ar131)unc-1(e538)
was significantly increased as compared with that in
unc-1(e538) (Fig. 3, A and B). The
number of apoptosis in unc-1(e538), examined as a control in
this study, was approximately the same as that in wild-type N2 (Fig.
3B). We next constructed
ced-3(n1286)sel-12(ar131) and
ced-4(n1894)sel-12(ar131)
mutants to clarify the mechanism of
sel-12(ar131)-induced cell death. The
ced-3 product is homologous to the mammalian
interleukin-1
-converting enzyme, which is essential for the
execution of programmed cell death. The ced-4 product is a
homologue of Apaf-1 and activates ced-3. Either of the
ced-3 and ced-4 mutations eliminates programmed
cell death (27, 29, 30). The examination of
ced-3(n1286)sel-12(ar131)
and
ced-4(n1894)sel-12(ar131) embryos demonstrated that ced-3(n1286) and
ced-4(n1894) inhibited sel-12(ar131)-induced apoptosis completely
(Fig. 3, B and C), which means that
sel-12(ar131)-induced apoptosis is dependent on
ced-3 and ced-4 activity.

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Fig. 3.
A, apoptotic cell corpses in each
strain. The Nomarski image of a representative embryo from each mutant
is shown. Cell corpses are indicated by arrowheads.
a, unc-1(e538); b,
sel-12(ar131)unc-1(e538).
B, quantitative analysis of N2,
unc-1(e538),
sel-12(ar131)unc-1(e538),
ced-3(n1286)sel-12(ar131)unc-1(e538),
and
ced-4(n1894)sel-12(ar131)unc-1(e538)
embryos. Embryos in the comma stage were scored for apoptotic cell
corpses.
sel-12(ar131)unc-1(e538)
showed an increased number of apoptosis compared with other mutants.
Also, ced-3(n1286) and
ced-4(n1894) inhibit apoptosis completely. *,
p < 0.01 versus
unc-1(e538). C, representative
Nomarski images of
ced-3(n1286)sel-12(ar131)
and ced-4(n1894)sel-12(ar131) embryos
are shown. Apoptosis is completely inhibited in these embryos.
a,
ced-3(n1286)sel-12(ar131);
b,
ced-4(n1894)sel-12(ar131).
|
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Dantrolene Treatment Prevents sel-12(ar131)-induced
Apoptosis--
Previous studies have indicated that perturbed
Ca2+ release from the ER is a critical factor for
FAD-related PS1-induced apoptosis (13, 31-33). Thus, we treated
sel-12(ar131)unc-1(e538)
with dantrolene, which specifically inhibits Ca2+ release
from the ER stores. In embryos of dantrolene-treated sel-12(ar131)unc-1(e538),
apoptosis was significantly decreased (Fig.
4), which suggests that, by abnormal
regulation of calcium release from the ER,
sel-12(ar131) predisposes embryo cells to apoptosis.

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Fig. 4.
Effects of dantrolene against
sel-12(ar131)-induced
apoptosis. Age-synchronous matured hermaphrodites were treated
with dantrolene to prevent Ca2+ release from the ER and
reduce intracellular Ca2+. The embryos were then collected
and scored for cell corpses. The number of apoptosis in
sel-12(ar131)unc-1(e538)
embryos was significantly reduced by dantrolene treatment.
*, p < 0.01 versus without
dantrolene.
|
|
 |
DISCUSSION |
This study demonstrated the role of
sel-12, a PS1 homologue of C. elegans, in
apoptosis depending on oxidative stress from mitochondrial dysfunction.
The sel-12(ar171) mutant, which genetically represents a null allele, confers increased resistance to paraquat and
hydrogen peroxide. To investigate the role of sel-12 in
apoptotic activities under oxidative stress from mitochondrial
dysfunction, apoptosis in sel-12mev-1 mutant embryos was
examined. The mev-1 gene encodes succinate dehydrogenase
cytochrome b large subunit (Cyt-1), which is a component of
complex II of the mitochondrial electron transport chain (34). This
mutation elevated the generation of reactive oxygen species from
mitochondria (21) and increased the number of apoptosis as compared
with wild-type N2 embryos.2 The
sel-12(ar171)mev-1(kn1)
mutant showed a decreased number of apoptosis as compared with
mev-1(kn1), which means that
sel-12(ar171) prevents mev-1-induced
apoptosis. This is consistent with our findings that
sel-12(ar171) is resistant to such oxidative
stress agents as exogenous paraquat or hydrogen peroxide.
On the other hand, in another allele mutant,
sel-12(ar131) significantly increased the number
of apoptosis as compared with that in wild type. Because the
supernumerary apoptotic cells were suppressed in
ced-3(n1286)sel-12(ar131)
and
ced-4(n1894)sel-12(ar131) embryos, this mean that the sel-12(ar131)-induced
apoptosis was ced-3- and
ced-4-dependent.
sel-12(ar131) happens to have a conserved point
mutation of PS1 C92S, which was reported by an Italian FAD
family.3 Hence, it is
expected that sel-12(ar131) has properties in
common with the PS1 C92S mutation and with another FAD-linked mutant PS1. The PS1 C92S mutation also increased A
42 production as did the
other FAD-related PS1 mutations (35). Increased A
42 production and
induction of apoptosis by the FAD-related mutant PS are considered to
be the result of a gain of function property (10, 13). Accordingly, it
is quite likely that sel-12(ar131) has a gain of
proapoptotic function similar to that of the FAD-related mutant PS.
Some studies have demonstrated that FAD-related mutant PS may sensitize
neurons to apoptosis by perturbing intracellular calcium homeostasis
(13, 31-33). We found that dantrolene treatment suppressed sel-12(ar131)-induced apoptosis, which means that
by abnormal regulation of calcium release from the ER,
sel-12(ar131) predisposes embryo cells to apoptosis.
Our study with sel-12(ar171), which represents a
null allele, indicated that sel-12 might itself induce
apoptosis under oxidative stress and mitochondrial dysfunction, whereas
sel-12(ar131), which is thought to have a gain of
function property, induced apoptosis through abnormal calcium release
from the ER. The defective vulva phenotype of sel-12 mutants
is rescued by human PS1 (19), which suggests that the basic function is
conserved between sel-12 and PS1. Therefore, PS1 might also
be similarly involved in inducing cell death under oxidative stress or
mitochondrial function. The findings of our study on
sel-12-induced apoptosis related with oxidative stress would
help to further the understanding of the cell death mechanism in
Alzheimer's disease.
We thank Dr. I. Greenwald for providing
sel-12 mutants.
Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M212058200
The abbreviations used are:
FAD, familial
Alzheimer's disease;
ER, endoplasmic reticulum;
PS1, presenilin-1.
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