Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations.
ABSTRACT Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. Mutations in presenilins 1 and 2 (PS1 and PS2) account for approximately 40% of familial AD (FAD) cases. FAD mutations and genetic deletions of presenilins have been associated with calcium (Ca(2+)) signaling abnormalities. We demonstrate that wild-type presenilins, but not PS1-M146V and PS2-N141I FAD mutants, can form low-conductance divalent-cation-permeable ion channels in planar lipid bilayers. In experiments with PS1/2 double knockout (DKO) mouse embryonic fibroblasts (MEFs), we find that presenilins account for approximately 80% of passive Ca(2+) leak from the endoplasmic reticulum. Deficient Ca(2+) signaling in DKO MEFs can be rescued by expression of wild-type PS1 or PS2 but not by expression of PS1-M146V or PS2-N141I mutants. The ER Ca(2+) leak function of presenilins is independent of their gamma-secretase activity. Our data suggest a Ca(2+) signaling function for presenilins and provide support for the "Ca(2+) hypothesis of AD."
- SourceAvailable from: Paolo Pinton[Show abstract] [Hide abstract]
ABSTRACT: Significance: in all cells, the endoplasmic reticulum (ER) and mitochondria are physically connected to form junctions termed mitochondria-associated membranes (MAMs). This subcellular compartment is under intense investigation because it represents a "hot spot" for the intracellular signaling of important pathways, including the synthesis of cholesterol and phospholipids, calcium homeostasis and reactive oxygen species (ROS) generation and activity. Recent advances: the advanced methods currently used to study this fascinating intracellular microdomain in detail have enabled the identification of the molecular composition of MAMs and their involvement within different physiopathological contexts. Critical issues: here we review the knowledge regarding i) MAMs composition in terms of protein composition, ii) the relationship between MAMs and ROS, iii) the involvement of MAMs in cell death programs with particular emphasis within the tumor context, iv) the emerging role of MAMs during inflammation and v) the key role of MAMs alterations in selected neurological disorders. Future directions: whether alterations in MAMs represent a response to the disease pathogenesis or directly contribute to the disease has not yet been unequivocally established. In any case, the signaling at the MAMs represent a promising pharmacological target for several important human diseases.Antioxidants & redox signaling. 01/2015;
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ABSTRACT: In this review we describe the present knowledge about store operated Ca2 + entry (SOCE) in neurons and the proteins involved in this process: STIM, as well as Orai and TRP channels. We address the issue of whether SOCE is used only to refill Ca2 + in the ER or whether Ca2 + that enters the neuronal cell during SOCE also performs signaling functions. We collected the data indicating that SOCE and its components participate in the important processes in neurons. This has implications for identifying new drug targets for the treatment of brain diseases. Evidence indicates that in neurodegenerative diseases Ca2 + homeostasis and SOCE components become dysregulated. Thus, different targets and strategies might be identified for the potential treatment of these diseases. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 01/2015; 740. · 5.30 Impact Factor
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ABSTRACT: One of the most critical pathological features of Alzheimer's disease (AD) is the accumulation of β-amyloid (Aβ) peptides that form extracellular senile plaques in the brain. Aβ is derived from β-amyloid precursor protein (APP) through sequential cleavage by β- and γ-secretases. γ-secretase is a high molecular weight complex minimally composed of four components: presenilins (PS), nicastrin, anterior pharynx defective 1 (APH-1), and presenilin enhancer 2 (PEN-2). In addition to APP, γ-secretase also cleaves many other type I transmembrane (TM) protein substrates. As a crucial enzyme for Aβ production, γ-secretase is an appealing therapeutic target for AD. Here, we summarize current knowledge on the structure and function of γ-secretase, as well as recent progress in developing γ-secretase targeting drugs for AD treatment.Frontiers in Cellular Neuroscience 01/2014; 8:427. · 4.18 Impact Factor
Presenilins Form ERCa2+LeakChannels,
a Function Disrupted by Familial
Alzheimer’s Disease-Linked Mutations
Huiping Tu,1Omar Nelson,1Arseny Bezprozvanny,1Zhengnan Wang,1Sheu-Fen Lee,2
Yi-Heng Hao,2Lutgarde Serneels,3Bart De Strooper,3Gang Yu,2and Ilya Bezprozvanny1,*
1Department of Physiology
2Center for Basic Neuroscience
UT Southwestern Medical Center at Dallas, Dallas, TX, 75390, USA
3Neuronal Cell Biology and Gene Transfer, Center for Human Genetics, Flanders Interuniversity Institute for
Biotechnology (VIB4) and KU Leuven, 3000 Leuven, Belgium
Alzheimer’s disease (AD) is a progressive and
irreversible neurodegenerative disorder. Muta-
tions in presenilins 1 and 2 (PS1 and PS2)
account for ?40% of familial AD (FAD) cases.
lins have been associated with calcium (Ca2+)
signaling abnormalities. We demonstrate that
wild-type presenilins, but not PS1-M146V and
PS2-N141IFAD mutants,canform low-conduc-
tance divalent-cation-permeable ion channels
in planar lipid bilayers. In experiments with
PS1/2 double knockout (DKO) mouse embry-
onic fibroblasts (MEFs), we find that presenilins
account for ?80% of passive Ca2+leak from the
in DKO MEFs can be rescued by expression of
wild-type PS1 or PS2 but not by expression of
PS1-M146V or PS2-N141I mutants. The ER
Ca2+leak function of presenilins is independent
of their g-secretase activity. Our data suggest
vide support for the ‘‘Ca2+hypothesis of AD.’’
Alzheimer’s disease (AD) is a neurodegenerative disorder
that currently affects nearly 2% of the population in indus-
tion of AD cases (familial AD, FAD) are characterized by an
lin 1 (PS1) and presenilin 2 (PS2) account for about 40% of
all known FAD cases (Tandon and Fraser, 2002). Preseni-
lins are 50 kDa proteins that contain nine transmembrane
domains (Laudon et al., 2005) and reside in the endoplas-
mic reticulum (ER) membrane (Annaert et al., 1999). The
complex of presenilins with nicastrin, aph-1, and pen-2
subunits functions as g-secretase, which cleaves the
amyloid precursor protein (APP) and releases the amyloid
b-peptide (Ab), the principal constituent of the amyloid
plaques in the brains of AD patients. Consistent with the
role of presenilins as catalytic subunits of g-secretase
(De Strooper et al., 1998; Wolfe et al., 1999), FAD muta-
tions in presenilins affect APP processing.
In addition to changes in APP processing, FAD muta-
tions in presenilins result in deranged calcium (Ca2+)
signaling (reviewed in Smith et al., 2005). What is a mech-
anistic explanation of these findings? Do presenilins
play a direct role in Ca2+signaling? Here we establish
that presenilins function as passive ER Ca2+leak channels
and that the FAD mutations of presenilins affect their abil-
ity to conduct Ca2+ions. Obtained results provide a new
insight into normal physiological function of presenilins
and strengthen the emerging connection between de-
ranged neuronal Ca2+signaling and AD (Khachaturian,
1989; LaFerla, 2002; Mattson et al., 2000; Smith et al.,
Recombinant Presenilins Form Cation-Permeable
Channels in Planar Lipid Bilayers
The predicted structure of presenilins includes nine trans-
membrane domains (Figure 1A), consistent with potential
ion-channel or transporter function. We proposed that
presenilins may mediate Ca2+transport in cells. To test
this hypothesis, we expressed human PS1 protein and
PS1-M146V and PS1-DE9 FAD mutants in Sf9 cells by
baculoviral infection. We also investigated PS1-D257A
(Figure 1A), a mutant that abolishes g-secretase activity
of presenilins (Wolfe et al., 1999). The efficient expression
of PS1, PS1-M146V, PS1-D257A, and PS1-DE9 was
confirmed by Western blotting (Figure 1B). The heterolo-
gously overexpressed presenilins are not incorporated
Cell 126, 981–993, September 8, 2006 ª2006 Elsevier Inc. 981
into the g-secretase complex and are mostly present as
holoproteins (Figure 1B). The uncleaved form of endoge-
nous presenilins is also present in untransfected mamma-
lian cells, including neurons (Annaert et al., 1999).
To investigate a potential ion-transport function of pre-
senilins, we adapted a planar lipid bilayer (BLM) reconsti-
tution technique (Tu et al., 2005a, 2005b). The ER micro-
somes from PS1-infected Sf9 cells were isolated and
fused with BLM. Ba2+ions (50 mM on the trans side)
were used in these experiments as a current carrier. We
did not detect ion currents across the BLM prior to fusion
of ER microsomes (Figure 2A, first column, n = 59) or fol-
lowing fusion of microsomes from noninfected Sf9 cells
(Figure 2A, second column, n = 11). In contrast, when mi-
crosomes from PS1-infected Sf9 cells were fused to the
BLM, currents were observed in 7 out of 9 experiments
(Figure 2A, third column). When compared to currents at
0 mV holding potential, the PS1-supported currents
were increased at ?10 mV holding potential and reduced
at +10 mV holding potential (Figure 2A, third column), in
agreement with the electrochemical gradient for Ba2+
ions. These data suggested that PS1 is able to facilitate
transport of Ba2+ions across the BLM, consistent with
the Ca2+channel function in vivo. In contrast to experi-
ments with PS1 microsomes, much smaller currents
were observed in experiments with PS1-M146V micro-
somes (Figure 2A, fourth column). The PS1-D257A mutant
supported Ba2+currents across the BLM similar to wild-
type PS1 (Figure 2A, fifth column), and the PS1-DE9 mu-
tant displayed enhanced channel function (Figure 2A,
sixth column). By analogy with other known ion channels,
the functional PS1 channels are likely to be multimers of
several PS1 subunits. Can the PS1:PS1-M146V multimer
function as an ion channel? To answer this question, we
coinfected Sf9 cells with PS1 and PS1-M146V baculovi-
ruses (in a 1:1 ratio), isolated ER microsomes, and fused
them to planar lipid bilayers. We did not detect channel
activity in these experiments (Figure 2A, seventh column),
suggesting that the PS1-M146V mutant exerts a domi-
nant-negative effect on ion-channel function of PS1.
Ca2+channels in the absence of divalent cations. Thus, in
the next series of experiments, we repeated BLM recon-
stitution experiments using 100 mM Cs+on the trans
side of the membrane. Cs+ions were used in these exper-
iments to minimize currents via endogenous potassium
channels present in ER microsomes. The results of bilayer
experiments obtained using Cs+as a current carrier were
consistent with results obtained using Ba2+, but the cur-
rents were ?4 times larger in the amplitude (see Fig-
ure S1A in the Supplemental Data available with this
PS1 and PS2 are two highly homologous mammalian
presenilin isoforms. Does PS2 share ion-channel function
Figure 1. Expression of Presenilins in Sf9
(A) Molecular model of presenilins (based on
Laudon et al., 2005). The transmembrane do-
mains (TM1–TM9), locations of aspartate resi-
dues critical for g-secretase activity, and the
site of endoproteolytic cleavage are indicated.
Positions of PS1-M146V, PS1-DE9, PS1-
D257A, and PS2-N141I mutations are shown.
(B and C) Expression of PS1, PS2, and mutants
in Sf9 cells. Microsomes prepared from nonin-
with PS1 and PS2 baculoviruses as indicated
were analyzed by Western blotting with anti-
PS1 (B) and anti-PS2 (C) monoclonal anti-
982 Cell 126, 981–993, September 8, 2006 ª2006 Elsevier Inc.
with PS1? To answer this question, we generated baculo-
virus encoding human wild-type PS2 and the PS2-N141I
FAD mutant (Figure 1A) and confirmed efficient expres-
sion of these proteins in Sf9 cells by Western blotting
(Figure 1C). We found that recombinant PS2, but not re-
combinant PS2-N141I, supported Ba2+and Cs+currents
in the BLM (Figure 2B; Figure S1B).
Channels Formed by Presenilins
Have Very Low Conductance
opening events in our experiments, and, instead, PS-
mediated currents had a ‘‘noisy’’ appearance (Figure 2).
These types of currents are typical for channels with
very low conductance, such as, for example, ICRACCa2+
Figure 2. PS1 and PS2 Form Small-Conductance Cation Channels in Planar Lipid Bilayers
the zero level for the current traces.For eachexperiment, 10 s of continuous current recording is shown. Similar results were obtained in at least three
experiments with each construct.
(C and D) The unitary current estimates are shown for experiments with 50 mM Ba2+(C) or 100 mM Cs+(D) in the trans compartment as mean ± SD
(n = number of independent BLM experiments, shown above each bar).
Cell 126, 981–993, September 8, 2006 ª2006 Elsevier Inc. 983
currents (Zweifach and Lewis, 1993). To estimate the size
of the unitary current via PS-supported channels, we
applied a modification of the stationary noise analysis
technique (see the Supplemental Data for details). By ap-
plying this method, we estimated that, at 0 mV, the size of
the unitary Ba2+currents supported by PS1 was equal to
0.04 ± 0.01 pA (n = 4); that is, ?50 times lower than the
size of the unitary current via InsP3R1 (2 pA) observed in
identical recording conditions (Tu et al., 2005b). In the
same conditions, the size of the unitary current was esti-
mated to be equal to 0.06 ± 0.02 pA (n = 3) for PS1-
D257A, 0.09 ± 0.02 pA (n = 3) for PS1-DE9, and 0.03 ±
0.01 pA (n = 4) for PS2 (Figure 2C). The size of the unitary
Cs+currents at 0 mV was estimated to be equal to 0.18 ±
0.04 pA (n = 4) for PS1, 0.21 ± 0.05 pA (n = 4) for PS1-
D257A, 0.25 ± 0.07 pA (n = 3) for PS1-DE9, and 0.09 ±
0.05 pA (n = 3) for PS2 (Figure 2D). Thus, the size of the
Cs+unitary current was 3- to 5-fold larger than the size
of the Ba2+unitary current for the wild-type PS1, wild-
type PS2, and the mutants that have been tested. In addi-
tional experiments, we performed recordings of PS1-
mediated channel activity using 200 mM Cs+as a current
tion led to a significant increase in the size of the observed
currents but that the unitary channel openings still could
not be resolved (Figure S2). By noise analysis, we esti-
mated the size of the unitary currents supported by PS1
in experiments with 200 mM Cs+to be equal to 0.51 ±
0.09 pA (n = 3).
To determine the single-channel conductance of PS1-
formed channels, we used noise analysis to estimate the
size of microscopic unitary currents at ?20 mV, ?10 mV,
0 mV, and +10 mV transmembrane voltages. From these
results, we determined that the average microscopic
conductance with Ba2+as a current carrier (gBa) is equal
to 1.2 pS for PS1, 0.002 pS for PS1-M146V, and 2.6 pS
for PS1-DE9 (Figure S3A). With Cs+as a current carrier,
the average microscopic conductance (gCs) is equal to
5.2 pS for PS1, 0.7 pS for PS1-M146V, and 8.3 pS for
PS1-DE9 (Figure S3B).
Purified PS1 Forms Cation Channels in the Bilayers
Is the presenilin protein itself or some additional protein or
proteins present in the Sf9 cell microsomal preparation re-
sponsible for the channel activity observed in the BLM
(Figure 2)? To answer this question, we generated baculo-
viruses encoding amino-terminal His-tagged PS1 and
PS1-M146V proteins. The microsomes from Sf9 cells in-
fected with His-PS1 and His-PS1-M146V baculoviruses
were solubilized in 1% CHAPS and used for purification
of His-PS1 and His-PS1-M146V proteins on an Ni-NTA
agarose column as previously described (Shah et al.,
2005). The purified His-PS1 and His-PS1-M146V proteins
lipids, ergosterol, and nystatin. The resulting proteolipo-
somes were highly enriched for His-PS1 and His-PS1-
M146V proteins (Figures 3A and 3B). Obtained liposomes
were used for planar lipid bilayer reconstitution experi-
ments performed in the presence of NaCl on both sides
of the membrane. Consistent with the previous report
(Woodbury and Miller, 1990), fusion of ergosterol/nysta-
tin-containing liposomes with the bilayer resulted in the
appearance of large transient currents (Figure 3C). Perfu-
sion of the cis chamber terminated fusion of liposomes
activity due to reduction in local ergosterol concentration
(Figure 3C). When the remaining currents were analyzed,
we observed Na+currents across the bilayer in experi-
ments with His-PS1 liposomes in 5 out of 7 experiments
(Figure 3D, third column). In contrast, no significant cur-
rent activity was observed in experiments with protein-
free liposomes (Figure 3D, second column, n = 5) and
with His-PS1-M146V liposomes (Figure 3D, fourth col-
umn, n = 4). The noise analysis performed as described
above yielded an average microscopic conductance
(gNa) equal to 5 pS for His-PS1 liposomes and 0.003 pS
for His-PS1-M146V liposomes (Figure S4).
Ca2+Signaling Defects in Presenilin Double-
Knockout (DKO) Mouse Embryonic Fibroblasts
To determine the physiological role of Ca2+channels
experiments with fibroblasts derived from presenilin dou-
ble-knockout mice (Herreman et al., 2000) (DKO cells).
Control experiments were performed with fibroblasts de-
rived from wild-type mice (MEF cells). In our experiments,
we loaded MEF and DKO cells with Fura-2 Ca2+imaging
dye and triggered InsP3R-mediated Ca2+release from
the ER by addition of 300 nM bradykinin (BK), an agonist
of PLC-coupled BK receptor. The cytosolic Ca2+concen-
tration in these experiments is calculated from the ratio
of Fura-2 signals at 340 nm and 380 nm excitation wave-
lengths shown by the pseudocolor images (Figure 4A).
Prior to stimulation with BK, cytosolic Ca2+was equal to
187 ± 41 nM (n = 28) in MEF cells and 135 ± 32 nM (n =
23) in DKO cells, significantly (p < 0.05) lower. Application
difference between the peak and the basal Ca2+levels
(D[Ca2+]) was equal to 225 ± 103 nM (n = 28) for MEF cells
and 787 nM ± 76 (n = 23) for DKO cells (Figure 4C). Thus,
the amplitude of BK-induced Ca2+responses was 3.5-
fold larger in DKO fibroblasts than in wild-type MEFs.
release in DKO cells? No significant changes in InsP3R1
expression levels were detected in DKO fibroblasts when
compared to MEFs by Western blotting (Figure S5). DKO
we reasoned that the DKO and MEF cells may differ in the
filling state of ER Ca2+stores. To compare the levels of
Ca2+stored in the ER, in the next series of experiments,
we evaluated Ca2+signals induced in MEF and DKO cells
by application of 5 mM ionomycin. Ionomycin is an iono-
phore that induces formation of Ca2+-permeable pores in
cellular membranes, leading to complete emptying of ER
984 Cell 126, 981–993, September 8, 2006 ª2006 Elsevier Inc.
Ca2+stores independently from the InsP3R activation. We
found that application of ionomycin resulted in more mas-
sive and longer-lasting Ca2+signals in DKO cells than in
MEF cells (Figure 4D). To estimate total ER Ca2+content
fromthese data, weintegrated the area under the ionomy-
average, the area under the curve was equal to 30 ± 6
mM3s (n = 25) for MEF cells and 55 ± 13 mM3s (n = 30)
for DKO cells (Figure 4E). Thus, the size of the ionomy-
cin-sensitive Ca2+pool is 1.8-fold larger in DKO cells
in DKO cells.
These results led us to propose that presenilins act as
ER Ca2+leak channels that facilitate passive Ca2+leak
across the ER membrane. To test this hypothesis more di-
rectly, we evaluated Ca2+signals induced in MEF and
DKO cells by thapsigargin. Application of thapsigargin
blocks SERCA Ca2+pump activity, leading to passive
ER Ca2+leak pathway. We found that application of 1 mM
thapsigargin caused rapid and large cytosolic Ca2+eleva-
tion in MEF cells, but much smaller and delayed cytosolic
Ca2+elevation in DKO cells (Figure 4F). On average, we
found that cytosolic Ca2+elevation could be detected at
16 ± 4 s (n = 17) after application of thapsigargin to MEF
cells and at 157 ± 14 s (n = 19) after application of thapsi-
gargin to DKO cells. To estimate the rate of endogenous
Ca2+leak in these experiments, we measured a slope of
cytosolic Ca2+increase in thapsigargin-exposed cells (re-
lease rate). We found that, on average, the release rate
was equal to 1.85 ± 0.24 nM/s (n = 17) in MEF cells and
0.33 ± 0.05 nM/s (n = 19) in DKO cells (Figure 4G). Thus,
genetic deletion of presenilins resulted in a 5.6-fold reduc-
tion in the rate of Ca2+leak across the ERmembrane, sug-
gesting that in MEF fibroblasts, presenilins account for
?80% of the total endogenous ER Ca2+leak activity.
Rescue of Ca2+Signaling Defects in DKO Fibroblasts
The defects in Ca2+signaling observed in DKO fibroblasts
periments. In these experiments, DKO fibroblasts were
transfected with EGFP plasmid alone (EGFP control) or
structs and analyzed by Fura-2 Ca2+imaging. In the first
Figure 3. Purified His-PS1 Forms Na+
Channels in Planar Lipid Bilayers
(A and B) Proteoliposomes containing purified
His-PS1 and His-PS1-M146V proteins were
analyzed by SDS gel electrophoresis followed
by Coomassie staining (A) or Western blotting
(C) Nystatin/ergosterol-mediated fusion of His-
PS1 proteoliposomes with BLM. Large nystatin
channels are observed upon fusion of proteoli-
posomes with the bilayer. The activity of nysta-
tin channels is terminated following perfusion
of the cis chamber.
(D) Na+currents are shown for empty BLM
(BLM) and for experiments with protein-free li-
posomes and proteoliposomes containing pu-
rifiedHis-PS1 and His-PS1-M146V. The dotted
lines represent the zero level for the current
with Na+as a current carrier (600 mM NaCl cis
and 150 mM NaCl trans). For each experiment,
10 s of continuous current recording is shown.
Similar results were obtained in at least three
experiments with each batch of liposomes.
Cell 126, 981–993, September 8, 2006 ª2006 Elsevier Inc. 985
series of experiments, DKO fibroblasts were transfected
with EGFP, EGFP + PS1, and EGFP + PS1-M146V con-
structs (Figure 5A). Under resting conditions, the average
basal Ca2+levels were equal to 183 ± 24 nM (n = 7) for
EGFP-transfected cells, 296 ± 28 nM (n = 12) for EGFP +
PS1-transfected cells, and 194 ± 42 nM (n = 17) for
0.05) higher in EGFP + PS1-transfected cells than in cells
of 300 nM BK induced large and transient Ca2+signals in
EGFP-transfected and EGFP + PS1-M146V-transfected
Figure 4. Ca2+Signaling in Wild-Type and Presenilin DKO Fibroblasts
(A) Representative Fura-2 images of bradykinin (BK) inducedCa2+responsesinMEF(top)andDKO(bottom)cells. 340/380Fura-2 ratiosareshownat
time points indicated. The pseudocolor calibration scale for 340/380 ratios is shown on the right. Three hundred nanomolar BK was added at t = 0.
(B) The time course of BK-induced [Ca2+] changes in MEF (red) and DKO (black) representative cells is shown.
(C) The average amplitude of BK-induced Ca2+release from MEF and DKO cells is shown as mean ± SD (n = number of cells).
(D) The time course of ionomycin (IO) induced [Ca2+] changes in MEF (red) and DKO (black) representative cells is shown.
(E) The average size of the ionomycin-releasable Ca2+pool is shown for MEF and DKO cells as mean ± SD (n = number of cells).
(F) The time course of thapsigargin (Tg) induced [Ca2+] changes in MEF (red) and DKO (black) representative cells is shown.
(G) The average slope of thapsigargin-induced Ca2+elevation is shown for MEF and DKO cells as mean ± SD (n = number of cells). The data from at
least three independent experiments with BK, IO, and Tg were combined for analysis. The average amplitude of BK-induced Ca2+response, the av-
erage size of the IO-releasable Ca2+pool, and the average rate of Tg-induced Ca2+leak are significantly (***p < 0.05) different in DKO cells compared
to MEF cells.
986 Cell 126, 981–993, September 8, 2006 ª2006 Elsevier Inc.
difference between the peak and basal Ca2+levels
(D[Ca2+]) in BK-stimulated cells was equal to 702 ± 82
nM (n = 7) for EGFP-transfected cells, 258 ± 92 nM
(n = 12) for EGFP + PS1-transfected cells, and 633 ± 125
nM (n = 17) for EGFP + PS1-M146V-transfected cells
To interpret these results, we reasoned that expression
of wild-type PS1 rescues the endogenous Ca2+leak path-
way in DKO cells and reduces intraluminal Ca2+levels and
the amount of released Ca2+. The increase in resting Ca2+
levels observed in PS1-transfected cells (Figures 5A and
5B) can also be explained by increased ‘‘leakiness’’ of
ER Ca2+stores under resting conditions. The longer dura-
tion of the BK-induced Ca2+transient in PS1-transfected
cells (Figures 5A and 5B) most likely reflects the fact that
the SERCA Ca2+pump requires more time to pump re-
leased Ca2+back into the ‘‘leaky’’ stores. Our BLM recon-
stitution experiments demonstrated that Ca2+channel
function is impaired in the PS1-M146V FAD mutant (Fig-
ure 2A). Consistent with these observations, expression
of the PS1-M146V mutant in DKO cells had no significant
effect on resting Ca2+levels or the amplitude and duration
of BK-induced Ca2+response (Figures 5A–5C), presum-
ably because the PS1-M146V mutant was not able to
facilitate Ca2+leak from the ER.
To test these ideas more directly, we evaluated the size
of the ionomycin-sensitive Ca2+pool in transfected DKO
cells. We found that addition of 5 mM ionomycin induced
a large and long-lasting elevation of cytosolic Ca2+levels
cells, but a smaller in amplitude and shorter in duration
Ca2+elevation in EGFP + PS1-transfected cells (Figure
5D). On average, the area under the Ca2+curve was equal
to 46 ± 9 mM3s (n = 19) for EGFP-transfected cells, 26 ± 7
mM3s (n = 21) for cells transfected with EGFP + PS1, and
48 ± 11 mM3s (n = 27) for cells transfected with EGFP +
PS1-M146V (Figure 5E). Thus, expression of wild-type
PS1 resulted in a 1.8-fold reduction in ER Ca2+content in
DKO cells, whereas expression of the PS1-M146V mutant
had no significant effect.
As an additional test of our hypothesis, we evaluated
thapsigargin-induced Ca2+signals in transfected DKO
cells. We found that application of 1 mM thapsigargin re-
sulted in rapid and massive cytosolic Ca2+elevation in
EGFP + PS1-transfected cells but delayed and much
smaller Ca2+elevation in EGFP-transfected and EGFP +
PS1-M146V-transfected cells (Figure 5F). On average,
we found that cytosolic Ca2+elevation could be detected
at 17 ± 3 s (n = 11) after application of thapsigargin to
DKO cells transfected with EGFP + PS1, at 133 ± 17 s
(n = 11) for DKO cells transfected with EGFP, and at 148 ±
18 s (n = 10) for DKO cells transfected with EGFP + PS1-
M146V. Further analysis revealed that the average rate of
thapsigargin-induced Ca2+leak was equal to 1.0 ± 0.2
nM/s (n = 11) for EGFP-transfected DKO cells, 5 ± 1 nM/
s (n = 11) for DKO cells transfected with EGFP + PS1,
and 0.99 ± 0.16 nM/s (n = 10) for DKO cells transfected
sive Ca2+leak is 5-fold higher in EGFP + PS1-transfected
cells than in cells transfected with EGFP alone or with
EGFP + PS1-M146V. Our results support the hypothesis
that the wild-type PS1, but not the PS1-M146V mutant,
is able to rescue ER Ca2+leak function impaired in DKO
In the next series of experiments, we utilized the same
paradigm to evaluate the ability of other presenilin expres-
sionconstructsto rescueCa2+signaling defects observed
in DKO cells. We found that expression of PS1-DE9 and
PS1-D257A mutants in DKO cells reduced the amplitude
of BK-induced Ca2+signals (Figure 6A) and the content
of the ionomycin-sensitive Ca2+pool (Figure 6B), similar
to expression of wild-type PS1. Expression of wild-type
PS2 reduced the amplitude of BK-induced Ca2+signals
(Figure 6A), but the PS2-N141I mutant was ineffective
(Figure 6A). Cotransfection of PS1 and PS1-M146V plas-
mids had no effect on BK-induced Ca2+signals (Fig-
ure 6A). Thus, we concluded that the behavior of all prese-
nilin constructs and construct combinations in DKO
fibroblast rescue experiments (Figure 6A) is consistent
with the behavior observed for the same constructs in
BLM reconstitution experiments (Figure 2). To rule out
potential artifacts resulting from transient overexpression
of PS1 and PS2, we performed a series of Ca2+imaging
experiments with stably transfected DKO fibroblasts.
stable lines was confirmed by Western blotting (Figure
S6). We found that the content of ionomycin-sensitive
Ca2+stores was reduced to wild-type levels in DKO cells
stably transfected with PS1 (HPS1 line), PS1-D257A,
PS1-DE9, or PS2 (HPS2 line), but not with PS2-N141L
The ability of the PS1-D257A mutant to rescue BK- and
ionomycin-induced Ca2+responses in DKO cells (Figures
6A and 6B) suggested that g-secretase function of PS1 is
dispensable for Ca2+signaling function studied in our ex-
periments. To further explore this issue, we performed
Ca2+imaging experiments with MEFs generated from
Aph-1abc?/?triple knockout mice and control Aph-1a+/+
(Aph-1bc?/?) MEFs. In separate experiments, we demon-
strated that g-secretase activity is completely absent in
oprotein inthesecellsatlevelssimilar tothosein wild-type
MEFs (L.S. and B.D.S., unpublished data). In experiments
with ionomycin, we found that the size of the ER Ca2+pool
was similar to that in wild-type MEFs for both Aph-1a+/+
and Aph-1abc?/?cells (Figure 6B).
Presenilins and ER Ca2+Homeostasis
The results obtained above are consistent with a role of
presenilins as passive ER Ca2+leak channels. As an addi-
tional test of this hypothesis, we performed a series of
Ca2+flux measurements with isolated ER microsomes.
In these experiments, Ca2+accumulated in microsomes
as a result of Ca2+pump activity and then was released
Cell 126, 981–993, September 8, 2006 ª2006 Elsevier Inc. 987
Figure 5. Rescue of Ca2+Signaling Defects in DKO Fibroblasts
(A) Representative Fura-2 images of BK-induced Ca2+responses are shown for DKO fibroblasts transfected with EGFP (first row), EGFP + PS1 (sec-
ond row), and EGFP + PS1-M146V (third row) expression plasmids. The transfected cells were identified by GFP imaging (first column). The data are
presented and analyzed as explained in the Figure 4 legend.
(B) The time course of BK-induced [Ca2+] changes in representative DKO cells transfected with EGFP (black), EGFP + PS1 (red), and EGFP + PS1-
M146V (blue) constructs is shown.
(C) The average amplitude of BK-induced Ca2+release from DKO cells transfected with EGFP, EGFP + PS1, and EGFP + PS1-M146V constructs is
shown as mean ± SD (n = number of cells).
(D) The time course of IO-induced [Ca2+] changes in representative DKO cells transfected with EGFP (black), EGFP + PS1 (red), and EGFP + PS1-
M146V (blue) constructs is shown.
(E) The average size of the IO-sensitive Ca2+pool is shown for DKO cells transfected with EGFP, EGFP + PS1, and EGFP + PS1-M146V constructs as
mean ± SD (n = number of cells).
(F) The time course of Tg-induced [Ca2+] changes in representative DKO cells transfected with EGFP (black), EGFP + PS1 (red), and EGFP + PS1-
M146V (blue) constructs is shown.
988 Cell 126, 981–993, September 8, 2006 ª2006 Elsevier Inc.
via a passive leak mechanism following addition of thapsi-
gargin. We discovered that the rate of thapsigargin-in-
duced Ca2+leak was equal to 0.41 ± 0.06 nM Ca2+/s
(n = 3) for microsomes from noninfected Sf9 cells, 1.1 ±
0.24 nM Ca2+/s (n = 5) for PS1-containing microsomes,
and0.35 ±0.07nMCa2+/s(n=3)for PS1-M146V-contain-
ing microsomes (Figure S7). In complementary experi-
ments, we found that the rate of thapsigargin-induced
Ca2+leak was equal to 1.82 ± 0.32 nM Ca2+/s (n = 5) for
MEF microsomes, 0.45 ± 0.09 nM Ca2+/s (n = 4) for DKO
microsomes, and 1.51 ± 0.27 nM Ca2+/s (n = 4) for HPS1
microsomes (Figure S8).
To further test our hypothesis, we directly measured
Ca2+concentration in the ER ([Ca2+]ER) of wild-type and
DKO MEFs with the low-affinity Ca2+imaging dye Mag-
Fura-2 (Hofer, 1999). Consistent with our expectations,
we found that [Ca2+]ERis elevated ?2-fold in DKO cells
(Figure 7A). On average, [Ca2+]ERwas equal to 87 ± 16
mM (n = 20) in MEF cells and 190 ± 48 mM (n = 28) in DKO
cells (Figure 7B). The transient transfection of DKO cells
on [Ca2+]ER(Figure 7B). In contrast, transfection of DKO
cells with PS1, PS1-D257A, or PS1-DE9 constructs re-
duced [Ca2+]ERto wild-type levels (Figure 7B). Consistent
with transient transfection results, [Ca2+]ERwas reduced
to wild-type levels in HPS1, PS1-D257A, PS1-DE9, or
HPS2 cell lines but remained elevated in PS2-N141L cells
(Figure 7B). [Ca2+]ERin both Aph-1a+/+and Aph-1abc?/?
MEFs was the same as in wild-type MEFs (Figure 7B), fur-
for proper control of ER Ca2+homeostasis.
Presenilins as ER Ca2+Leak Channels
What determines a steady-state level of intraluminal Ca2+
in the ER? The calculations predict that, under physiolog-
ical conditions, the SERCA pump reaches thermodynam-
ical equilibrium when intraluminal [Ca2+]ERis equal to 2.4
rect imaging of intraluminal [Ca2+]ERresulted in estimates
in the 100–500 mM range (Hofer, 1999) (Figure 7A). The
most likely explanation for this difference is the leakiness
of the ER membrane for Ca2+ions. According to this idea
(Figure 7C, left panel), the steady-state ER intraluminal
Ca2+level is determined by an equilibrium between
SERCA-mediated movement of Ca2+from the cytosol
into the ER lumen and the passive leak of Ca2+from ER
into the cytosol. Experimental estimates for the rate of
Ca2+leak vary from 19 mM ER Ca2+/min in acinar cells to
90 mM ER Ca2+/min in neurons (Camello et al., 2002).
A number of candidates have been previously consid-
ered to play a role as ER Ca2+leak channels, such as the
ribosome-translocon complex (Lomax et al., 2002; Van
Coppenolle et al., 2004), the antiapoptotic protein bcl-2
(Pinton et al., 2000), and InsP3R (Oakes et al., 2005). How-
(G) The average slope of Tg-induced Ca2+elevation is shown for DKO cells transfected with EGFP, EGFP + PS1, and EGFP + PS1-M146V constructs
as mean ± SD (n = number of cells).
The data from at least three independent experiments for each experimental condition were combined for analysis. The average amplitude of
BK-induced Ca2+response, the average size of the IO-releasable Ca2+pool, and the average rate of Tg-induced Ca2+leak are significantly
(***p < 0.05) different in DKO cells transfected with EGFP + PS1 compared to DKO cells transfected with EGFP or EGFP + PS1-M146V.
Figure 6. Summary of DKO Fibroblast Rescue Experiments
(A) The average amplitude of BK-induced Ca2+release from DKO cells
shown as mean ± SD (n = number of cells analyzed). When compared
to DKO cells transfected with EGFP alone, the amplitude of BK-in-
duced Ca2+response is significantly (***p < 0.05) smaller in DKO cells
transfected with EGFP + PS1, EGFP + PS1-DE9, EGFP + PS1-D257A,
and EGFP + PS2.
(B) The average size of the IO-sensitive Ca2+pool is shown for DKO
cells transiently transfected with EGFP and PS1 rescue constructs
and for stably transfected DKO cells as mean ± SD(n = number of cells
analyzed). When compared to DKO cells transfected with EGFP alone,
the size of the IO-releasable Ca2+pool is significantly (***p < 0.05)
smaller in DKO cells transfected with EGFP + PS1, EGFP + PS1-
DE9, and EGFP + PS1-D257A. The size of the IO-releasable Ca2+
pool is also significantly (***p < 0.05) smaller in HPS1, PS1-D257A,
PS1-DE9, and HPS2 cell lines.
Cell 126, 981–993, September 8, 2006 ª2006 Elsevier Inc. 989
remains an ‘‘enigma of Ca2+signaling’’ (Camello et al.,
2002). Prior to endoproteolytic cleavage, presenilins are
localized to the ER membrane (Annaert et al., 1999). Here
we propose that the holoprotein form of presenilins func-
tions as an ER Ca2+leak channel in cells (Figure 7C, left
panel). This hypothesis is consistent with the ability of
PS1 and PS2 proteins to form low-conductance divalent-
cation-permeable channels in the BLM (Figure 2) and
with Ca2+signaling abnormalities in DKO cells (Figure 4).
Moreover, [Ca2+]ERin DKO cells is 2-fold higher than in
wild-type MEFs (Figure 7A), directly supporting our hy-
pothesis. Importantly, cytosolic and ER Ca2+signaling de-
fects in DKO fibroblasts can be rescued by expression of
PS1 and PS2 constructs (Figure 5, Figure 6, and Figure 7).
From these results, we conclude that, in MEF fibroblasts,
presenilins account for ?80% of ER Ca2+leak activity.
Notably, our conclusions differ from the recent study of
Ca2+signaling in DKO MEFs performed by a different
methodology (Kasri et al., 2006).
alytic subunit of the g-secretase complex (De Strooper
et al., 1998; Wolfe et al., 1999). Here we propose that pre-
senilins also serve as ER Ca2+leak channels. Moreover,
our results indicate that g-secretase function of preseni-
lins is dispensable for ER Ca2+leak function. In our exper-
iments, the catalytic mutant PS1-D257A formed channels
in the bilayers (Figure 2) and rescued all Ca2+signaling de-
fects in DKO cells (Figure 6 and Figure 7). Moreover, the
filling state of ER Ca2+stores was normal in Aph-1abc?/?
MEF cells (Figure 6B and Figure 7B), which completely
lack g-secretase activity (Serneels et al., 2005). Interest-
ingly, only the ‘‘mature’’ (cleaved) form of presenilins can
Figure 7. Presenilins and ER Ca2+Homeostasis
(A) Representative ER Ca2+traces recorded by ER-loaded Mag-Fura-2 in wild-type (red line) and DKO (black line) MEFs. Cells were loaded with Mag-
Fura-2 and permeabilized by 10 mM digitonin in buffer containing 170 nM Ca2+and 3 mM ATP. At the end of the experiment, the ER membrane was
permeabilized with 5 mM ionomycin in the Ca2+-free buffer. 340/380 Mag-Fura-2 ratios were converted to [Ca2+]ER.
(B) The average ER Ca2+concentration determined as shown in (A) is presented for wild-type MEF cells, DKO cells transiently transfected with EGFP
and PS1 rescue constructs, and stably transfected DKO rescue cells as mean ± SD (n = number of cells). When compared to DKO cells transfected
with EGFP alone, the ER Ca2+concentration is significantly (***p < 0.05) lower in DKO cells transfected with EGFP + PS1, EGFP + PS1-D257A, and
EGFP + PS1-DE9. The ER Ca2+concentration is also significantly (***p < 0.05) smaller in HPS1, PS1-D257A, PS1-DE9, and HPS2 stable lines.
(C) Model of Ca2+homeostasis in wild-type and PS mutant cells. In wild-type cells (left), steady-state ER intraluminal Ca2+levels are determined by
N141I FAD mutants, ER Ca2+leak function of presenilins is impaired, resulting in higher steady-state intraluminal Ca2+levels and lower cytosolic Ca2+
levels. Following generation of InsP3and opening of InsP3R, the amplitude of Ca2+response is higher in mutant cells (right) than in wild-typecells (left)
due to the larger driving force for Ca2+ions exiting from the ER to the cytoplasm.
990 Cell 126, 981–993, September 8, 2006 ª2006 Elsevier Inc.
function as g-secretase (Tandon and Fraser, 2002),
whereas in our experiments, ER Ca2+leak function was
supported by the holoprotein form of presenilins.
Deranged Ca2+Signaling and AD
Ca2+signaling defects have been observed in studies with
fibroblasts from FAD patients (Ito et al., 1994), in FAD cel-
lularand animal models (LaFerla, 2002; Smith etal., 2005),
Anumber of hypotheses have been previously considered
to explain these phenomena. It has been proposed that
Ab-peptides associate into small aggregates that form
Ca2+-permeable channels in the plasma membrane of
neurons, causing Ca2+influx (Arispe et al., 1993). Another
hypothesis states that the APP intracellular domain
(AICD) released by g-secretase cleavage affects Ca2+sig-
naling by regulating expression of neuronal Ca2+signaling
proteins (Leissring et al., 2002). A correlation between the
PS1-M146V mutation and enhanced function of neuronal
ryanodine receptors has been recently described (Stutz-
mann et al., 2006). Here we propose that PS1-M146V
and PS2-N141I FAD mutations in presenilins affect neuro-
nal Ca2+signaling by disrupting the ER Ca2+leak pathway
(Figure 7C, right panel). Our model is supported by the in-
lent-cation-permeable channels in the BLM (Figure 2) and
the failure of these mutants to rescue cytosolic and ER
Ca2+signaling defects in PS1/2 DKO MEFs (Figure 5, Fig-
ure 6, and Figure 7). When coexpressed with the PS1-
M146V mutant, wild-type PS1 did not form cation-perme-
able channels in BLM (Figure 2A) and failed to restore the
amplitude of BK-induced Ca2+release in DKO fibroblasts
(Figure 6A). Thus, we propose that PS1-M146V and PS2-
N141I mutants exert a dominant-negative effect on PS-
supported ER Ca2+leak function, resulting in intraluminal
ER Ca2+overload at the steady state (Figure 7C, right
panel). We further propose that ER Ca2+overload in cells
expressing PS1-M146V and PS2-N141I mutants leads to
supranormal Ca2+release following activation of InsP3R
(Figure 7C, right panel). The proposed model (Figure 7C)
is consistent with increased InsP3-induced Ca2+release
in Xenopus oocytes expressing PS1-M146V and PS2-
N141I FAD mutants (Leissring et al., 1999a, 1999b,
2001), in synaptosomes and cortical neurons from PS1-
M146V mutant knockin mice (Begley et al., 1999; Stutz-
mann et al., 2004), and in hippocampal neurons from
PS2-N141I transgenic mice (Schneider et al., 2001). It is
highly likely that the abnormal intraluminal ER Ca2+levels
predicted by our model (Figure 7C) may also be responsi-
ble for Ca2+signaling defects observed in cells from PS1
and PS2 knockout mice (Herms et al., 2003; Ris et al.,
2003; Takeda et al., 2005; Yoo et al., 2000).
In our bilayer experiments, we found that the PS1-DE9
FAD mutant had increased ER Ca2+leak channel activity
when compared to wild-type PS1 (Figure 2). These results
suggest that PS1-DE9 is a gain-of-function ER Ca2+leak
mutant, whereas PS1-M146V and PS2-N141I are domi-
nant-negative ER Ca2+leak mutants. The gain-of-function
Ca2+leak phenotype of the PS1-DE9 mutant is consistent
with an earlier observation of elevated basal Ca2+levels in
SH-SY5Y cells transfected with PS1-DE9expression con-
struct (Cedazo-Minguez et al., 2002).
normal Ca2+signaling has been compiled in a recent
review article (Smith et al., 2005). In addition to PS1-
M146V/L, PS2-N141I/L, and PS1-DE9, these mutants
also include PS1-H163R, PS1-A246Q/E, PS1-L286V,
and PS2-M239V/I (Smith et al., 2005). It will be interesting
to evaluate ER Ca2+leak function of these FAD mutants.
The ‘‘loss of presenilin function’’ hypothesis of FAD was
put forward based on the analysis of age-dependent neu-
rodegeneration in PS1/2 cDKO mice (Saura et al., 2004).
Our observation of loss of ER Ca2+leak channel function
in PS1-M146V and PS2-N141I mutants is in general
agreement with the ‘‘loss of presenilin function’’ hypothe-
sis of FAD (Saura et al., 2004). Additional studies will also
be required to investigate the connection between defec-
tive Ca2+signaling induced by PS FAD mutations, abnor-
mal g-secretase activity, and neurodegeneration in AD.
Expression Constructs and Recombinant Baculoviruses
The following human PS1 and PS2 expression constructs were gener-
ated in pFastBac1 baculovirus vector (Invitrogen) and pcDNA3 mam-
malian expression vector (Invitrogen): PS1 = M1-I467, PS1-M146V,
PS1-D257A, PS1-DE9 (M1-I467, del T291–S319); PS2 = M1-I448,
PS2-N141I. Recombinant baculoviruses were generated using the
Bac-to-Bac system (Invitrogen) as previously described (Shah et al.,
2005; Tu et al., 2005a, 2005b). Expression of presenilins in Sf9 cells
was confirmed by Western blotting with anti-PS1 (MAB5232, Chemi-
con) and anti-PS2 mAb. The samples used for Western blotting were
maintained at 37?C prior to loading on the gel.
Planar Lipid Bilayer Experiments
Planar lipid bilayer (BLM) recordings of PS-supported currents were
performed as previously described for studies of InsP3R (Tu et al.,
2005a, 2005b) (see Supplemental Data for details). The ion currents
across the BLM were amplified (OC-725C, Warner Instruments), fil-
tered at 5 kHz, digitized (Digidata 1200, Axon Instruments), and stored
on a computer hard drive and recordable optical discs. For presenta-
tion, the current traces were digitally filtered at 200 Hz (pClamp 6.0,
Axon Instruments). For offline computer analysis, the stationary noise
analysis method was used (see Supplemental Data for details).
Purification and Reconstitution of His-PS1
His-PS1 and His-PS1-M146V baculoviruses were generated as de-
scribed above. The recombinant His-PS1 and His-PS1-M146V
proteins were solubilized in 1% CHAPS and purified by affinity chro-
matography on Ni-NTA agarose (Shah et al., 2005). The purified His-
PS1 and His-PS1-M146V proteins were reconstituted into liposomes
composed of phospholipids, ergosterol, and nystatin and used in the
planar lipid bilayer experiments by following published procedures
(Woodbury and Miller, 1990) (see Supplemental Data for details).
MEF Ca2+Imaging Experiments
DKO and MEF fibroblasts cultured on poly-D-lysine (Sigma) coated 12
mm round glass coverslips were established as described (Herreman
et al., 2000). Cytosolic Ca2+imaging experiments with DKO and MEF
Cell 126, 981–993, September 8, 2006 ª2006 Elsevier Inc. 991
(MSN) primary cultures (Tang et al., 2003) (see Supplemental Data for
details). In rescue experiments, DKO cells were transfected using Lip-
ofectamine (Invitrogen) with pEGFP-C3 plasmid (Clontech) or with
a 1:3 mixture of pEGFP-C3 and PS expression plasmids (in pcDNA3)
48 hr after transfection. The transfected cells were identified by GFP
imaging. The stable DKO rescue lines and Aph-1abc?/?MEF lines
were previously described (Nyabi et al., 2003; Serneels et al., 2005).
For ER Ca2+measurements with Mag-Fura-2 dye, we used proce-
dures previously described for BHK fibroblasts (Hofer, 1999) (see Sup-
plemental Data for details).
Microsomal Ca2+Flux Measurements
ER microsomes were isolated by gradient centrifugation from nonin-
fected Sf9 cells; from Sf9 cells infected with PS1 and PS1-M146 bacu-
loviruses; and from wild-type, DKO, and HPS1 MEFs. The isolated mi-
crosomes were used for Ca2+flux measurement with Fura-2 (see
Supplemental Data for details). The initial rate of thapsigargin-induced
Ca2+flux was measured for each type of microsome as explained in
Supplemental Data include Supplemental Experimental Procedures,
Supplemental References, and eight figures and can be found with
this article online at http://www.cell.com/cgi/content/full/126/5/981/
We are grateful to Donald Hilgemann for advice on thermodynamical
calculations and for comments on the paper, to Tie-Shan Tang for
advice on Ca2+imaging experiments, to Malu Tansey for helping
with DKO and MEF cells, to Tianhua Lei for help with Sf9 cell culture,
and to Janet Young for administrative assistance. I.B. is supported
by the Robert A. Welch Foundation, NINDS R01 NS38082, UT South-
western Medical Center Alzheimer’s Disease Center grant NIA P30
AG12300, and Alzheimer’s Association award IIRG-06-24703. G.Y. is
supported by the Robert A. Welch Foundation and NIA R01
AG023104. B.D.S. is supported by a Pioneer award from the Alz-
heimer’s Association, the KU Leuven (GOA 2004/12), and the Federal
Office for Scientific Affairs, Belgium (IUAP P5/19).
Received: June 1, 2005
Revised: April 9, 2006
Accepted: June 30, 2006
Published: September 7, 2006
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