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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 19, 2001 as doi:10.1096/fj.00-0541fje. |
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Department of Life Science, Kwangju Institute of Science and Technology, Puk-gu, Kwangju 500–712, Korea.
2Correspondence: Department of Life Science, Kwangju Institute of Science and Technology, 1- Oryong-dong, Puk-gu, Kwangju 500–712, Korea. E-mail: ykjung{at}eunhasu.kjist.ac.kr
SPECIFIC AIM
In this study we hypothesized that calsenilin/DREAM/KChIP3 could exert pro-apoptotic and amyloidogenic effects on cells. We have now assessed the effects of down-regulation or overexpression of calsenilin/DREAM/KChIP3 on apoptosis and the production of amyloid-ß peptide (Aß).
PRINCIPAL FINDINGS
1. Attenuation of apoptosis by down-regulation of endogenous
calsenilin/DREAM/KChIP3
We initially examined the contribution of calsenilin/DREAM/KChIP3
to apoptosis by directly targeting calsenilin/DREAM/KChIP3 expression
in Jurkat cells by using an anti-sense oligonucleotide (AS-5). Exposure
to calsenilin/DREAM/KChIP3 AS-5 effectively reduced apoptosis mediated
by A23187, a Ca2+-ionophore, and by thapsigargin,
an endoplasmic reticulum Ca2+-ATPase
inhibitor—both of which are known to increase the cytosolic free
Ca2+ concentration—and by Fas (Fig. 1A-C
). The highly significant
suppression of apoptosis was observed in cells exposed to A23187 (Fig. 1A)
. The diminished expression of calsenilin/DREAM/KChIP3 was
determined by Western blot analysis of Jurkat cells
treated with either AS-5 or scrambled oligonucleotides, which served as
a negative control.
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2. Induction of apoptosis by overexpression of
calsenilin/DREAM/KChIP3
Cells expressing calsenilin/DREAM/KChIP3 exhibited the
morphological and biochemical features of apoptosis. Determination of
the viability of GFP-positive cells based on their morphology showed
that ectopic expression of calsenilin/DREAM/KChIP3-GFP for 48 h
induced apoptosis in 52% of HeLa cells. The apoptosis was suppressed
effectively by the pan-caspase inhibitor z-VAD-fmk, which supports the
active role of caspases in calsenilin/DREAM/KChIP3-induced apoptosis.
The incidence of apoptosis declined to 29% with expression of a
truncated calsenilin/DREAM/KChIP3, which lacked the C-terminus
Ca2+ binding domain (
C-cal). Conversely, when
cells expressing calsenilin/DREAM/KChIP3 were exposed to 0.5 µM
A23187, the incidence of apoptosis increased to 64%. In addition, when
the extent to which Bcl-xL would suppress the
calsenilin/DREAM/KChIP3-induced apoptosis was tested, we found that
expression of Bcl-xL in HeLa
(HeLa/Bcl-xL) cells decreased the level of
apoptosis to 19%.
3. Calsenilin/DREAM/KChIP3 -mediated Aß42 formation
Given that expression of Swedish amyloid-ß precursor protein
mutant (APPsw) substantially increased the incidence of
calsenilin/DREAM/KChIP3-induced apoptosis (data not shown), we next
examined whether calsenilin/DREAM/KChIP3 exerted an effect on the
formation of Aß42 in B103/APPsw cells, rat neuroblastoma cells stably
expressing B103/APPsw. Quantitative determination of Aß42 peptide by
using a specific ELISA revealed that transfection of increasing amounts
of the pcDNA3.1-calsenilin/DREAM/KChIP3 plasmid resulted in
dose-dependent increases of Aß42 (Fig. 2A
); the amount of extracellular Aß42 peptide produced from
cells transfected with 3 µg of pcDNA3.1-calsenilin/DREAM/KChIP3 being
fourfold higher than that from cells transfected with the same amount
of control vector (pcDNA3.1) (Fig. 2A)
. Levels of exogenous expression
of calsenilin/DREAM/KChIP3 were then confirmed with Western blot
analysis. These results suggest that calsenilin/DREAM/KChIP3
contributes to the production of Aß42 peptide.
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4. Potentiation effect of C-terminus of PS2 on
calsenilin/DREAM/KChIP3-induced apoptosis and Aß42 formation
As calsenilin/DREAM/KChIP3 is known to bind to the
C-terminus of presenilin 2 (PS2), we examined whether the pro-apoptotic
activity of calsenilin/DREAM/KChIP3 is associated with PS2. B103/APPsw
cells were transiently transfected with
pEGFP-calsenilin/DREAM/KChIP3 and pcDNA3-HA-PS2 alone or in
combinations, and cell viability was determined. Expression of PS2
potentiated the incidence of calsenilin/DREAM/KChIP3-induced apoptosis
(from 52% to 72%) (Fig. 2B)
. In contrast, expression of PS2/411stop,
which is truncated at C-terminal 411 residue and does not colocalize
with calsenilin/DREAM/KChIP3 (data not shown), did not augment
calsenilin/DREAM/KChIP3-induced apoptosis, which indicates the pro-
apoptotic activity of calsenilin/DREAM/KChIP3 to be potentiated by the
C-terminus of PS2.
To determine whether our conclusion regarding the potentiation effect
of PS2 on calsenilin/DREAM/KChIP3-induced apoptosis is applicable to
Aß formation, we examined the secretion of Aß42 from B103/APPsw
cells transient expressing PS2 or PS2/411stop in the presence of
calsenilin/DREAM/KChIP3. Consistent with the results of Figure 2B
, expression of PS2 augmented the incidence of
calsenilin/DREAM/KChIP3-induced Aß42 secretion, but PS2/411stop did
not (Fig. 2C)
.
CONCLUSIONS AND SIGNIFICANCE
Apoptotic cell death and increased production of amyloid-ß peptide (Aß) are pathological features of Alzheimer’s disease (AD), although the exact contribution of apoptosis to the pathogenesis of the disease remains unclear. Calsenilin/DREAM/KChIP3 was identified recently by different groups as a Ca2+-binding protein that binds to the C-terminus of PS1 and PS2, and to the A-type voltage-gated potassium channel, and that serves as a transcription factor known as downstream regulatory element antagonist modulator. Here we described a novel pro-apoptotic function of calsenilin/DREAM/KChIP3. By anti-sense oligonucleotide-induced inhibition of calsenilin/DREAM/KChIP3 synthesis, apoptosis induced by Fas, Ca2+-ionophore, or thapsigargin is attenuated. Conversely, calsenilin/DREAM/KChIP3 expression induced the morphological and biochemical features of apoptosis, and overproduction of Aß42 in cells expressing APPsw. In addition, calsenilin/DREAM/KChIP3-induced apoptosis was suppressed by caspase inhibitor z-VAD and by Bcl-xL, and was potentiated by expression of APPsw, PS2, or increasing cytosolic Ca2+, but not by a PS2/411stop. In addition, calsenilin/DREAM/KChIP3 expression increased Aß42 production in cells expressing APPsw, which was potentiated by PS2, but not by PS2/411stop, which suggests a role for apoptosis-associated Aß42 production of calsenilin/DREAM/KChIP3.
Previous studies demonstrated that neurons undergoing apoptosis also
have been shown to increase the rate of Aß formation by three- to
fourfold. We observed significant increase of apoptosis and Aß42 in
neuronal B103/APPsw cells expressing calsenilin/DREAM/KChIP3 (about
fourfold). Calsenilin/DREAM/KChIP3 may modulate 
-secretase activity
by interacting with PS. Several studies have suggested that wild-type
or the C-terminus of PS plays an important role in apoptosis and Aß42
production. It is interesting that ALG-3, which codes for the
calsenilin/DREAM/KChIP3-binding region spanning the cytoplasmic
C-terminal 103 amino acids of PS2, rescues T-cell hybridoma and PC12
cells from apoptosis—probably by antagonizing the pro-apoptotic
function of PS2. Moreover, modifications of amino acids at the
C-terminus of PS abrogated the ability of PS mutations to overproduce
Aß. We also found that calsenilin/DREAM/KChIP3 colocalized with PS2
within ER, but not with PS2/411stop (data not shown) and that
calsenilin/DREAM/KChIP3-induced apoptosis and Aß42 production was
augmented by PS2, but not by PS2/411stop. These findings implicate that
calsenilin/DREAM/KChIP3 may be one of the missing molecules linking
apoptosis and Aß42 production to the C-terminus of PS.
Our findings suggest that Ca2+, as a
Ca2+-binding protein, may modulate the
pro-apoptotic function of calsenilin/DREAM/KChIP3. Inhibition of
calsenilin/DREAM/KChIP3 synthesis effectively prevented
Ca2+-mediated apoptosis as shown in Figure 1
. In
addition, the incidence of apoptosis declined with expression of a
truncated calsenilin/DREAM/KChIP3 that lacked the
Ca2+-binding domain of the C-terminus (C-cal);
conversely, apoptosis increased in cells expressing
calsenilin/DREAM/KchIP3 exposed to a
Ca2+-ionophore. Further, in a number of
experimental models, PS and their mutations were linked to
perturbations in calcium signaling, which in turn were related to
increased production of peptide Aß.
In these contexts, our study provides a potential link between
apoptosis, PSs, Aß42 production, and Ca2+
homeostasis (Fig. 3
). In this model, overexpression of ALG-3 may lead to sequestration of
cytoplasmic calsenilin/DREAM/KChIP3, thereby limiting the
calsenilin/DREAM/KChIP3-PS interactions and suppressing apoptosis.
Our findings demonstrated the pro-apoptotic and amyloidogenic activites
of calsenilin/DREAM/KChIP3, which may be modulated by intracellular
calcium concentration and mediated by interacting with PS and a
potassium channel or by serving as a
Ca2+-regulated transcription factor. The fact
that K+ efflux has been suggested to contribute
to neuronal apoptosis raises a possibility that calsenilin/DREAM/KChIP3
mediates apoptosis by acting as a potassium channel regulator.
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Considering these observations, we suggest that calsenilin/DREAM/KChIP3 may function in the pathogenesis of AD by modulating apoptosis and Aß production, though more detailed mechanism of calsenilin/DREAM/KChIP3-mediated apoptosis remains to be elucidated.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0541fje To cite this article, use (January 19, 2001) FASEB J. 10.1096/fj.00-0541fje 
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