From the Department of Neuropathology,
Ludwig-Maximilians-Universität, 81377 Munich, Germany and the
¶ Experimental Genetics Group, Department of Human Genetics,
B-3000 Leuven, Belgium
Received for publication, July 8, 2002, and in revised form, October 29, 2002
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ABSTRACT |
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Mutant presenilin-1 (PS1) increases amyloid
peptide production, attenuates capacitative calcium entry (CCE), and
augments calcium release from the endoplasmatic reticulum (ER). Here we measured the intracellular free Ca2+ concentration in
hippocampal neurons from six different combinations of transgenic and
gene-ablated mice to demonstrate that mutant PS1 attenuated CCE
directly, independent of the expression of the amyloid precursor
protein (APP). On the other hand, increased Ca2+ release
from the ER in mutant PS1 neurons, as induced by thapsigargin, was
clearly dependent on the presence of APP and its processing by PS1,
i.e. on the generation of the amyloid peptides and the APP
C99 fragments. This observation was corroborated by the
thapsigargin-induced increase in cytosolic
[Ca2+]i in PS1 deficient neurons, which
accumulate C99 fragments due to deficient The formation of amyloid peptides (A Here, we have analyzed cellular mechanisms in neurons that were derived
from six different types of single and multiple transgenic or
gene-ablated mice. We analyzed, for the first time, neurons that
express a mutant PS1 on a wild-type and an APP-deficient background,
and in addition we analyzed the effect of a mutant APP expressed in
wild-type and in PS1-deficient neurons. First and foremost, we
demonstrate here that CCE is normal in the absence of APP, whereas APP
deficiency did not correct the attenuated CCE provoked by mutant PS1.
These findings implicate PS1 directly in CCE, independent of its
activity as Strains of Transgenic Mice--
The parent APP[V717I],
PS1[wt], and PS1[A246E] transgenic mice (7, 10, 11, 12) and
APP Neuronal Cell Cultures--
Hippocampi from newborn mice were
dissociated with papain (25 units/ml, with cysteine, 2 mg) in
Dulbecco's modified Eagle's medium for 60 min at 37 °C
before transfer to Dulbecco's modified Eagle's medium/fetal calf
serum (5%), N2 supplement, glutamine, and antibiotics.
Cells were triturated and, after attachment on wild-type astrocyte
feeder layers, the medium was changed to neurobasal medium with B27
supplement (Invitrogen). Neurons were cultured for 19-21 days,
and experiments were performed in Locke's buffer, i.e. 154 mM NaCl, 5.6 mM KCl, 2.3 CaCl2, 1.0 mM MgCl2, 3.6 mM NaHCO3, 5 mM glucose, and 5 mM
HEPES (pH 7.2). Dissociation of neurons from adult mouse hippocampi was
as described (7).
Calcium Imaging--
[Ca2+]i measurements
in hippocampal neurons from newborn mice and dissociated neurons from
adult mice were performed using the indicator fura-2-acetoxymethyl
ester (fura-2/AM; Molecular Probes, Göttingen, Germany).
Fura-2/AM was solubilized in Dulbecco's modified Eagle's medium with
pluronic acid (0,08%) in HBSS (145 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 20 mM HEPES, 10 mM glucose, 1.8 mM
CaCl2) containing bovine serum albumin (1%). Cells on
cover slips were loaded at 37 °C for 30 min and transferred after 30 min of washing time to a microchamber on the stage of an upright microscope (BX50 WI, Olympus) to be viewed under visible and UV light
using a 60× water immersion objective. Approximate intracellular Ca2+ concentrations ([Ca2+]i) were
calculated from the ratio of a fura-2 emission evoked by 340-and 380-nm
light from a 75-W argon lamp using a digital imaging system (Till
Photonics, Munich, Germany). Measurements were acquired at 1-3 s
intervals at both excitation wavelengths (340 and 380 nm). Digital
fluorescence images were constructed and displayed as pseudo-color
images and subsequently analyzed (Vision software, Till Photonics,
Munich, Germany). [Ca2+]i was calculated for each
pixel in the frame with fluorescence intensities over a defined
threshold (14). Each experiment was performed on at least four cultures
after 19-21 days in culture and prepared from different mice.
Experiments were performed at room temperature, and drugs were applied
by bath superfusion (2 ml/min). For Ca2+-free experiments
the same buffer was used, but CaCl2 was omitted and 50 µM EGTA was added.
[Ca2+]i measurements in hippocampal slices were
performed using patch clamp methodology on pyramidal cells in thin slices following standard procedures (15, 16). Briefly, the brain from
an adult mouse (6-8 months) was dissected, and the hippocampus was
rapidly isolated and kept in ice-cold bicarbonate-buffered saline
solution (125 mM NaCl, 1.25 mM KCl, 1.25 mM KH2PO4, 25 mM
NaHCO3, 2 mM CaCl2, 1.5 mM MgCl2, and 16 mM glucose).
Transverse vibratome slices (150 µm) (16, 17) were maintained for at least 1 h at 34 °C in the same buffered saline solution with
continuous oxygenation (95% O2, 5% CO2).
Slices were transferred to the recording chamber and superfused with
the same solution at room temperature. Pyramidal cells were selected
under the microscope using a 60× water-immersion objective. Electrodes
pulled from borosilicate glass capillaries were filled with loading
solution containing 125 µM Fura-2, 140 mM
CsCl2, 2 mM MgCl2, 4 mM
ATP, 0.4 mM GTP, and 10 mM HEPES (pH 7.3)
(pipette resistance 2-3 MV). Whole-cell recordings were performed with
a patch clamp amplifier (EPC-9, HEKA Electronic, Lambrecht, Germany).
Cells were voltage clamped at a slightly depolarizing holding potential
( Mutant PS1 Attenuates CCE Independent of Expression of
APP--
CCE was measured in neurons from non-transgenic mice in
parallel with neurons from transgenic mice expressing human mutant PS1[A246E] or human wild-type PS1 as an extra control. CCE was induced by a published protocol, i.e. after preincubation in
Ca2+-free media containing cyclopiazonic acid to deplete
the ER Ca2+ stores, and the neurons were replenished with
Ca2+-containing media (1.8 mM) (6, 20). CCE was
strongly decreased in PS1[A246E] neurons (Fig.
1, A and B) and, as
an extra control, we observed that under the same conditions SKF-96365
inhibited CCE in wild-type neurons (Fig. 1, C and
D).
We then observed that CCE was normal in neurons derived from APP
The combined results demonstrated that mutant PS1 reduced neuronal CCE
independent of the expression of APP, which in itself also had no
negative effect on CCE. This is the first demonstration that attenuated
CCE is a direct consequence of mutant PS1, whereas APP and its
proteolytic processing are not involved in this process. It is
therefore concluded that PS1 is not implicated in CCE as the alleged
Mutant PS1 Increases ER Calcium Stores Only in the Presence of
APP--
Because APP processing did not affect CCE, we examined
whether the same would hold true for the ER calcium ion stores, which are increased by mutant PS1 (7, 9). The analysis was performed by
measuring the response to thapsigargin in hippocampal neurons derived
from six different transgenic mouse stains, i.e. transgenic mice with mutant PS1 and/or mutant APP and in combination with a
deficiency in APP (13) or in PS1 (9) (Table
I).
First and foremost we observed that, similar to CCE, the absence of APP
did not markedly affect the extent or the kinetics of changes in
[Ca2+]i in cultured hippocampal neurons in
response to thapsigargin (Fig. 2,
C and D). On the other hand, and opposite to the
effect on CCE, the absence of APP completely abolished the marked
increase in [Ca2+]i observed in mutant PS1
neurons (Fig. 2, E and F).
This demonstrated that the absence of APP did not negatively affect
either neuronal CCE or the ER-based calcium ion stores. The results do
reveal, however, that mutant PS1 can only increase ER-based calcium
ions stores in the presence of APP, which evidently implicates APP
itself or more likely its proteolytically derived fragments in
this intracellular phenomenon.
Deficiency of PS1 Also Causes Overfilling of ER Calcium
Stores--
In dissociated hippocampal neurons expressing
APP[V717I], the peak [Ca2+]i response to
thapsigargin was marginally increased relative to neurons from
non-transgenic mice (Fig. 3, A
and B). The same mutant APP[V717I] expressed in
PS1-deficient neurons, however, dramatically increased the amplitude of
the response to thapsigargin (Fig. 3, C and D),
indicating that the absence of PS1 strongly augmented the effect of
mutant APP. This was also evident in PS1(n
These important observations were further corroborated in experiments
with hippocampal slice preparations from the same strains of transgenic
mice in which the fluorescent dye was loaded from the patch pipette
(17). Brain sections have the unmistakable advantage that the neurons
maintain their physiological context with many cell processes intact.
The basal levels of [Ca2+]i in neurons in the
sections were lower than in isolated neurons but very similar for all
genotypes analyzed (Fig. 4).
Most importantly, in response to thapsigargin,
[Ca2+]i was again most markedly enhanced in
hippocampal neurons in slice preparations from PS1(n
The concordant results demonstrated that the deficiency of PS1 on its
own did markedly affect the response to thapsigargin as an indication
of the size of ER-based calcium ion stores. The responses were further
augmented by coexpression of mutant APP. These data warrant the
conclusion that PS1 affected the ER-based calcium ion stores indirectly
and only by the intermediation of APP, which, considering the nature of
these proteins and their known relationship, implicates the proteolytic
processing products from APP as prime candidates (Table I) (9,
21).
Although the normal physiological functions of both APP and PS1
and their intricate relationship are still unclear and heavily debated,
the evidence that mutant presenilins generate more hydrophobic amyloid
peptides is accepted (for reviews, see Refs. 22-24). The final step in
the processing of APP and the generation of the amyloid peptides is
Disturbed neuronal calcium ion homeostasis could play a subordinate but
also an essential role in the neurodegeneration that causes AD (for
reviews, see Refs. 2 and 25-27). Indications of the nature of the
alterations, caused either by PS1 itself or by APP cleavage products,
was analyzed by us and others in relevant paradigms, i.e.
neurons and brain sections derived from transgenic mice (5, 6, 7, 21).
We have now addressed these questions directly by comparatively
analyzing neurons derived from six different transgenic mouse strains
expressing either mutant PS1 or mutant APP on APP- or PS1-deficient
backgrounds (Table I).
First, we present experimental evidence that APP is not directly
involved in CCE or ER-based calcium ion storage, a finding with major
implications for the physiological function of APP. In contradiction to
results obtained in fibroblasts derived from APP In contrast to the direct attenuation of CCE by mutant PS1, we present
robust evidence that the increase of ER-based calcium stores by mutant
PS1 indeed requires the expression of APP. In the double transgenic
mice that express mutant PS1 on the APP It is clear that the thapsigargin-sensitive calcium ion movements in
mutant PS1 neurons are dependent on the presence of APP, leading to the
conclusion that this involves the The critical role of the C99 fragments is corroborated by the proposed
neurotoxicity of these fragments as demonstrated in cell biological
models (Refs. 2, 26, and references therein). Most recently, we have
obtained the first indications for the same effect in vivo
in adult mice that lack neuronal PS1 (9). It is not clear, however, how
the amyloid and C99 fragments disturb the ER-based calcium ion stores.
In transfected cells, the C57 fragment of APP appeared to induce
similar alterations in ER calcium stores as the C99 fragment (27). It
was proposed that the C57 fragments interact with the nuclear adaptor
protein Fe65, which, in turn, interacts with transcription factors (31,
32). This would mean that alterations in ER calcium storage in
PS1 transgenic mice are due to transcriptional effects of the C57
intracellular cleavage product of APP, i.e. the APP
intracellular domain (AICD), in complete analogy to the modes of
generation and action of the intracellular cleavage product of Notch
(NICD) (31). Although AICD was below detection limits in the brains of
the transgenic mice studied here (results not shown), we can evidently
not rule them out. Conversely, we observed increased ER calcium ion
storage in PS1 The most direct explanation of the similar results we obtained in
neurons that either express mutant PS1 or are deficient in PS1 would be
that the gain of function of mutant PS1 leads to increased levels of
the AICD or C57 fragments, whereas the loss of function of PS1
increases the C99 fragments. But then we must accept that the C99 and
the C57 fragments act similarly with respect to calcium homeostasis,
which could be at the transcriptional level. This requires additional
experimental definition of the mechanisms involved in both types of
genetically modified neurons.
The problem of whether and how alterations in neuronal calcium
homeostasis relate to loss of synaptic functions and eventually to
nerve cell loss in Alzheimer's disease has been speculated upon before
(for a review, see Ref. 2). Based on results from experiments performed
with fibroblasts (33) and lymphocytes (34) from AD patients carrying a
mutant PS1 gene, it was proposed that neurons in these patients might
suffer from alterations in ER calcium ion homeostasis well before the
patients develop the mental disorder. Alterations in intracellular free
calcium concentration have also been implicated in other
neurodegenerative diseases, i.e. prion disease (35) and
amyotrophic lateral sclerosis (36) as well as normal aging (37).
In conclusion, we analyzed neurons derived from the six most relevant
single and combined transgenic and gene-deficient mouse strains to
demonstrate that altered neuronal calcium dynamics caused by mutant PS1
are dual in nature. First, the hypothesis that mutant PS1 attenuates
CCE directly, independent of APP as proposed (6), is now proven by the
data presented here. Secondly, as opposed to CCE, the increase of
ER-calcium stores by mutant PS1 is found to be dependent on APP and is
correlated closely with the combination of changes in the levels of the
amyloid peptides and the C99 fragments of APP. How these fragments
alter ER-calcium stores is a matter of debate. The C99 fragments might
act either directly and similarly to the amyloid peptides, or they
might be further degraded and act like the APP intracellular domain AICD (27). Our observations do not lead to a complete understanding of
the normal physiological roles of APP and PS1, which are clearly much
more complex and diverse than anticipated from the pathological consequences of their malfunction. Conceivably, the pathogenic consequences of perturbed calcium homeostasis also offer possibilities for therapeutic approaches. Conversely, the possible benefits of using
-secretase activity.
Moreover, co-expression of mutant APP[V717I] in PS1-deficient neurons
further increased the apparent size of the ER calcium stores in
parallel with increasing levels of the APP processing products. We
conclude that mutant PS1 deregulates neuronal calcium homeostasis by
two different actions: (i) direct attenuation of CCE at the
cell-surface independent of APP; and (ii) indirect increase of
ER-calcium stores via processing of APP and generation of amyloid
peptides and C99 fragments.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
40/42) by proteolytic
processing of the amyloid precursor protein
(APP)1 is proposed to be
central to the pathology in Alzheimer's disease (AD) (1). The amyloid
peptides are neurotoxic and alter calcium ion homeostasis (for review,
see Ref. 2). Most familial forms of AD are caused by a mutation in
presenilin-1 (PS1) (for review, see 3). Mutant PS1 increases the
production of amyloid peptides by
-secretase cleavage of APP, an
activity for which PS1 is essential (4). In addition, however, mutant
PS1 deregulates intracellular calcium homeostasis by increasing the
apparent intracellular pools of Ca2+ and decreasing
capacitative calcium entry (CCE) (Refs. 5-7, and references therein).
CCE is a refill mechanism allowing entry of extracellular calcium ions
through plasma membrane channels that are tightly regulated by and even
physically linked to intracellular stores (for review, see Ref. 8).
Reduced CCE in neurons from mutant PS1 transgenic mice (5, 6) appeared
to increase the levels of A
42 in cultures, whereas exogenously added
peptide had no effect on CCE (6). The mechanisms by which mutant PS1 deregulates neuronal calcium homeostasis are not known, but the relation of a perturbed flux of calcium ions to the alteration in APP
processing, or vice versa, is debated or questioned as an
essential element in the pathogenic processes (5, 6, 7 and references therein).
-secretase on APP. We further measured and
compared the thapsigargin-induced increase in
[Ca2+]i as a measure of ER calcium stores in
neurons from the six different transgenic mouse strains, including mice
with a neuronal deficiency in PS1, i.e. PS1 (n
/
) crossed
with APP[V717I] mice (9). The results demonstrate that the increase
in ER calcium stores in neurons is due to increased processing of APP
into amyloid peptides and C99 fragments.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice (13) were described previously. Viable adult mice with a
specific neuronal deficiency in PS1, denoted PS1(n
/
), were
generated by crossing mice with a floxed PS1 gene with mice expressing
Cre recombinase under control of the mouse thy1 gene
promoter (9). PS1(n
/
) mice are viable and fertile and have
normal brain morphology and normal behavior (9). They were crossed with
APP[V717I] transgenic mice (10) to demonstrate effective inhibition
of amyloid peptide and plaque formation up to the age of 18 months
(9).
60 mV). Changes in [Ca2+]i were monitored
using a digital imaging system as described above. Following
establishment of the whole-cell configuration, loading of the cell with
fura-2 was monitored until equilibration between pipette and proximal
dendrite as indicated by stable maximal intensity signals at 340 nm.
Consecutive paired exposures to 340 and 380 nm were used to construct
background-corrected digital fluorescence images. The gain of the
intensified video CCD camera was set at values that optimized detection
of signals from the cell soma, and [Ca2+]i was
calculated (14). The calibration constants Keff (effective binding constant) and Rmin,
i.e. fluorescence ratio at zero Ca2+, were
obtained from in vivo calibration experiments as described (18, 19). These parameters varied slightly in time, depending on
several factors such as aging of the UV lamp used for excitation, but
typical values for Keff,
Rmin, and Rmax were 420, 0.4 and 1.4 nM, respectively. All patch clamp measurements
were performed at room temperature.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Attenuation of CCE by mutant PS1 is not
dependent on expression of APP. A and B,
decreased CCE in hippocampal neurons from PS1[A246E] transgenic mice
(n = 30 cells) as opposed to normal CCE in neurons from
non-transgenic mice and human wild-type (WT) PS1 transgenic
mice (*, p < 0.05). C and D,
cultured hippocampal neurons from non-transgenic and APP /
mice have
similar CCE, and CCE is inhibited by SKF-96365 (SKF; 100 µM). Data points are mean ± S.E. (n = 40 cells) (**, p < 0.001). E and
F, decrease in CCE is similar in neurons from PS1[A246E]
and APP
/
× PS1[A246E] mice (*, p < 0.05).
[Ca2+]i was measured by ratiometric imaging of
fura-2 in primary cultures of hippocampal neurons as illustrated in
Fig. 2A.
/
mice (Fig. 1, C and D), excluding endogenous APP
as a direct or indirect mediator of the effect of mutant PS1. We
further analyzed CCE in neurons from double transgenic mice expressing the mutant PS1 on an APP-deficient background, i.e. APP
/
× PS1[A246E]. It must be noted that the APP
/
× PS1[A246E]
mice were normally viable and fertile and did not show phenotypic or
histological abnormalities of the brain (results not shown). Most
importantly, however, we observed that CCE in the APP
/
× PS1[A246E] neurons was very similar to that in PS1[A246E] neurons,
i.e. in both CCE was significantly suppressed relative to
non-transgenic or APP
/
neurons (Fig. 1, E and
F).
-secretase but via another inherent or associated activity.
Overview of the six transgenic mouse strains analyzed with the most
relevant characteristics
and
C99 levels refer to the total levels of amyloid peptides and C99
fragments of APP, respectively, in the brains of the transgenic mice as
published (7-12). The symbols used to denote the
(semi)-quantitative changes as discussed in the text are: =, not
significantly different;
, absent; N.D., not determined; + and
,
increased or decreased, respectively, proportional with the number of
signs.
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Fig. 2.
ER-calcium stores are normal in APP /
neurons and increased in PS1[A246E] neurons. A and
B, a representative experiment of
[Ca2+]i measurements by ratiometric imaging of
fura-2 in primary cultures of hippocampal neurons. The time course of
fluorescence ratio (340/380 nm), with time points indicated
(panel A), of digital fluorescence images (panel
B) of a fura-2 loaded pyramidal cell is shown. The ratio
signal in panel A was calculated over the area outlined in
section B1 of panel B. C
and D, response to thapsigargin (Th) is similar
in hippocampal neurons from wild-type mice (WT) and
APP-deficient mice (APP
/
). E and F,
correction by APP deficiency of the increased response of
[Ca2+]i in PS1[A246E] neurons triggered by
thapsigargin. All data points represent mean fluorescence ratio ± S.E. (n = 40-50 cells) (*, p < 0.05).
Thapsigargin (1 µM) was applied as indicated by the
bars under the tracings in panels A,
C, and E.
/
) neurons that react to
thapsigargin by a robust and significant increase in cytoplasmic
calcium ion levels, although less pronounced than APP[V717I] × PS1(n
/
) neurons (Fig. 3, C and D) (Table
I).
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Fig. 3.
Increased ER-calcium stores in PS1(n /
)
and APP[V717I] × PS1(n
/
) neurons.
[Ca2+]i was measured by ratiometric imaging of
fura-2 (see Fig. 2 and "Experimental Procedures" for details).
A and B, increased response to thapsigargin
(Th) of [Ca2+]i in PS1[A246E]
neurons (*, p < 0.05) and the non-significant effect
in APP[V717I] neurons. C and D, increased
response to thapsigargin of [Ca2+]i in
PS1-deficient neurons (PS1(n
/
) (*, p < 0.01) is
strongly accentuated by the expression of APP[V717I] (**,
p < 0.005). Thapsigargin (1 µM) was
applied as indicated by the bars under the tracings
(panels A and C). Data points are mean
values ± S.E. of 40-55 cells from at least three independent
preparations.
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Fig. 4.
Increased ER-calcium stores in hippocampal
sections from PS1(n /
) and from APP[V717] × PS1(n
/
) mice. A, digital micrographs of a
hippocampal pyramidal cell in an acute slice preparation loaded with
fura-2 by a patch pipette as described under the "Experimental
Procedures." The images are at the different time points indicated in
the 340/380 nm fluorescence ratio tracing, calculated over the area
indicated in section A1 of panel A. B and C, response to thapsigargin (Th)
of [Ca2+]i in APP[V717I] neurons is not
significantly different from that in wild-type neurons, whereas the
increase in PS1-deficient neurons (PS1(n
/
) (*, p < 0.01) is strongly accentuated by the expression of APP[V717I] (**,
p < 0.001). Thapsigargin was applied by superfusion (2 ml min
1) as indicated by the bar (panel
B). Data points in panel B are mean fluorescence
ratios ± S.E. (n = 50-55 cells), whereas in
panel C the peak values of [Ca2+]i are
given (*, p < 0,05; **, p < 0,001).
/
) × APP[V717I] mice (Fig. 4, B and C) relative to
APP[V717I] neurons, which showed a marginal increase (Fig.
4C). Remarkably again, in preparations from PS1(n
/
) mice the increase in [Ca2+]i response to thapsigargin
was significant (Fig. 4C), confirming the results obtained
in cultured hippocampal neurons (Fig. 3).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-secretase cleavage, for which PS1 is essential (4). The primary
pathogenic changes in AD have been attributed to the amyloid peptides,
and different mechanisms have been proposed to explain their effect on
neurons and their role in neurodegeneration and dementia.
/
mice (27), we did
not observe any alterations in cultured hippocampal neurons from
APP
/
mice in response to thapsigargin or bradykinin. By analyzing
CCE in double transgenic mice that express the mutant PS1 on an
APP
/
background, we observed that the attenuation of CCE by mutant
PS1 is not dependent on the expression of APP (Table I). The
attenuation of CCE by mutant PS1 must then be due to a direct
interference with the mechanism of CCE that is independent of APP and
APP-cleavage and hence independent of the
-secretase activity of
PS1. The conclusion that a normal physiological function of PS1 is to
be sought in the regulation of CCE and the refilling of the ER-calcium
stores could be structurally explained if PS1 participates in the
formation of a channel within the plasma and/or ER-membrane that
mediates calcium ion movements. This proposition finds strong support
in the fact that PS1 is present at the cell surface (28), which was
most recently and convincingly confirmed and functionally extended to
other partners, i.e. nicastrin (29). The implication of a
close and intricate structural relationship of the plasma and ER
membranes in neurons to the function of neuronal signaling and calcium
homeostasis as well as in terms of the possible contribution to the
molecular basis of memory and neurodegeneration was proposed and
discussed extensively (Refs. 8, 25, 30, and references therein). There is clearly a need for the experimental demonstration that PS1
takes part in a novel structural entity linking the plasma and the ER
membranes to establish the molecular identity of the proposed
structures now diversely known as "plasmerosomes" or "store-operated channels" (Refs. 8, 25, 30, and references therein). Evidently in this respect as in many others, neurons are
structurally and functionally much more complex and differently organized than fibroblasts or other simple cell types. This may explain
the divergent results we obtained here in APP
/
neurons relative to
APP
/
fibroblasts (27).
/
background, the deficit in
ER calcium signaling was restored to normal values. This result
evidently excludes the possibility that APP-dependent
alterations in ER calcium signaling are responsible for the depression
of CCE by mutant PS1, as proposed (5). The argument
that higher levels of ER calcium ions in PS1 mutant cells would impair
CCE by preventing the agonists from depleting the intracellular calcium
stores beyond a threshold required to activate CCE (5) is contradicted
by our finding that CCE is still attenuated by mutant PS1 in the
APP
/
double transgenic mice, whereas the filling of ER-calcium
stores is not altered in the absence of APP (Table I).
-secretase activity of PS1 and
therefore the proteolytical fragments of APP, i.e. amyloid
peptides and their obligatory precursors, the C99 fragments. The
analysis of PS1-deficient neurons yielded, to our surprise, similar
alterations in ER calcium storage as observed in mutant PS1 neurons.
Because amyloid peptide production is very low in PS1
/
neurons (4,
9), we must conclude that the C99 fragments mediated the observed
alterations of the ER calcium signaling. The relative changes in the
size of the ER-calcium ion stores sensitive to thapsigargin correlate
very well with the relative changes in the level of the amyloid and C99
fragments in the brains of the mice analyzed (Table I, right
panel) (9-12).
/
neurons in which the generation of AICD is strongly inhibited. This contradicts results obtained by others in fibroblasts (27) and primary neurons derived from conventional PS1-knockout mice
(6). Possible factors contributing to this discrepancy include the fact
that the latter PS1
/
neurons were derived from embryonic tissues
from total PS1 knockout mice, whereas we derived primary neuronal
cultures from postnatal conditional PS1 knockout mice in which the
floxed PS1 gene is inactivated by coexpressed Cre recombinase driven by
the thy1 gene promoter (9). However, here we also analyzed
the response to thapsigargin in a different experimental setting,
i.e. in hippocampal slices derived from adult PS1(n
/
)
mice and demonstrated a very similar effect as in the postnatal primary
cultured neurons.
-secretase inhibitors for the treatment of AD are seriously challenged by the current and previous results, as these inhibitors will disturb neuronal calcium homeostasis by increasing the levels of
the N-terminal fragments of APP (9).
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FOOTNOTES |
---|
* This work was supported by the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen, the European Economic Community 5th Framework Program, the Rooms Fund, the KULeuven Research Fund, KULeuven R&D, and Deutsche Forschungsgemeinschaft Grant SFB 596.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
Postdoctoral fellow at Fonds voor Wetenschappelijk
Onderzoek- Vlaanderen.
** To whom correspondence should be addressed: Experimental Genetics Group, Dept. of Human Genetics, KULeuven-Campus Gasthuisberg ON_06, B-3000 Leuven Belgium. Tel.: 32-16-3458-88; Fax: 32-16-3458-71; E-mail: fredvl@med.kuleuven.ac.be.
Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M206769200
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ABBREVIATIONS |
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The abbreviations used are: APP, amyloid precursor protein; AD, Alzheimer's disease; PS1, presenilin-1; CCE, capacitative calcium entry; DFI, digital fluorescence images; AICD, APP intracellular domain.
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