From the Department of Neuropathology,
Ludwig-Maximilians-Universität, 81377 Munich, Germany,
¶ Laboratory of Neuroscience, University of Mons-Hainaut,
7000 Mons, Belgium,
Experimental Genetics Group,
Katholieke Universiteit Leuven, 3000 Leuven, Belgium, and the
§§ Janssen Research Foundation, 2340 Beerse,
Belgium
Received for publication, December 6, 2000, and in revised form, January 12, 2001
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ABSTRACT |
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Mutant human presenilin-1 (PS1)
causes an Alzheimer's-related phenotype in the brain of transgenic
mice in combination with mutant human amyloid precursor protein by
means of increased production of amyloid peptides (Dewachter, I., Van
Dorpe, J., Smeijers, L., Gilis, M., Kuiperi, C., Laenen, I.,
Caluwaerts, N., Moechars, D., Checler, F., Vanderstichele, H. & Van
Leuven, F. (2000) J. Neurosci. 20, 6452-6458) that
aggravate plaques and cerebrovascular amyloid (Van Dorpe, J., Smeijers,
L., Dewachter, I., Nuyens, D., Spittaels, K., van den Haute, C.,
Mercken, M., Moechars, D., Laenen, I., Kuipéri, C., Bruynseels,
K., Tesseur, I., Loos, R., Vanderstichele, H., Checler, F., Sciot, R. & Van Leuven, F. (2000) J. Am. Pathol. 157, 1283-1298). This gain of function of mutant PS1 is approached here in three paradigms that relate to glutamate neurotransmission. Mutant but not wild-type human PS1 (i) lowered the excitotoxic threshold for kainic acid in vivo, (ii) facilitated
hippocampal long-term potentiation in brain slices, and (iii)
increased glutamate-induced intracellular calcium levels in isolated
neurons. Prominent higher calcium responses were triggered by
thapsigargin and bradykinin, indicating that mutant PS modulates the
dynamic release and storage of calcium ions in the endoplasmatic
reticulum. In reaction to glutamate, overfilled Ca2+ stores
resulted in higher than normal cytosolic Ca2+ levels,
explaining the facilitated long-term potentiation and enhanced
excitotoxicity. The lowered excitotoxic threshold for kainic acid was
also observed in mice transgenic for mutant human PS2[N141I] and was
prevented by dantrolene, an inhibitor of Ca2+ release from
the endoplasmic reticulum.
Similar post-mortem diagnostic features in all Alzheimer's
disease (AD)1 patients,
sporadic or familial, suggest common pathogenic mechanisms. Dominant
early onset familial AD (EOFAD) is mainly caused by mutant presenilin1
(PS1) (3), which like mutant amyloid precursor protein (APP) increase
the production of amyloid peptides (A Amyloid deposits and neurofibrillary tangles are the obligatory
diagnostic lesions in all AD patients. This duality is not explained by
an increased production of A The profound repercussions for diagnosis and therapy of the exact
mechanisms in AD are evident. To study the pathological mechanism
in vivo, we generated transgenic mice that overexpress human
mutant PS. Very unlike humans, EOFAD mutant PS1 or PS2 transgenic mice
show no strong phenotype or pathological defects. Only in combination
with human APP do mutant PS increase the production of A We studied three paradigms that yield converging and convincing
evidence for disrupted neuronal calcium ion homeostasis by mutant PS.
The seizure threshold for the excitotoxin kainic acid (KA) was lower,
whereas neuronal damage in the hippocampus was prevented by dantrolene,
an inhibitor of calcium ion release from the ER. Standard hippocampal
LTP was normal in PS1 transgenic mice as opposed to APP transgenic mice
(26) but was facilitated when evoked by weak tetanic stimulation.
Finally, dynamic changes in intracellular [Ca2+] in
cultured neurons were disturbed by mutant PS1 by a mechanism that
involved the filling and release of Ca2+ from stores in the
ER, the cellular organelle where PS reside predominantly.
Transgenic Mice--
PS1 cDNA, wild-type or A246E mutant
(5), and wild-type or mutant N141I PS2 cDNA (27) were spliced in
the mouse thy-1 gene (28) to generate transgenic mice
by our standard methods (1, 2, 26, 28). Founders and offspring were
genotyped by polymerase chain reaction and Southern blotting of
tail-tip DNA. Transgenic strains were established and maintained in the FVB/N background and back-crossed to obtain mice homozygous for either
wild-type or mutant human PS1 or PS2.
Analysis of Expression--
Northern blots on total brain
mRNA were scanned and quantified (1, 26, 28). Western blots of
mouse brain extracts, i.e. in 5 mM Tris, 250 mM sucrose, 1 mM EGTA (pH 7.4) containing a
mixture of proteinase inhibitors. Homogenates were cleared (12,000 × g, 10 min, 4 °C) before membranes were pelleted by
high speed centrifugation (100,000 × g, 30 min,
4 °C). Membrane proteins were denatured and reduced, analyzed
on 4-20% Tris-glycine polyacrylamide gels (Novex, San Diego, CA), and
transferred to nitrocellulose filters. N- and C-terminal fragments of
PS1 and PS2 were detected with polyclonal antibodies B14/5, B17/2, and
B24/2 as described (1, 29).
Reactivity to Kainic Acid--
The doses of KA injected
intraperitoneally were established in preliminary experiments to elicit
seizures reproducibly with only moderate neuronal damage. Mice were
observed for 2 h postinjection, and the frequency and intensity of
seizures were rated in seven stages: lethargy, rigid posture, head
bobbing or circling, clonic seizure, rearing alone or with falling,
tonic-clonic seizures, and death. Pretreatment with dantrolene (10 mg/kg intraperitoneal) was for 30 min prior to injection of KA (30).
Histology for hippocampal damage was done 24 h after KA
injection on perfused and fixed brains (4% paraformaldehyde)
dehydrated and embedded in paraffin. Sections (7 µm) were stained
with hematoxylin-eosin and cresyl violet for hippocampal damage and
scored on a scale from 1 to 5 (31): 1 = minor damage to some
pyramidal cells in CA1 or CA3; 2 = mild damage to a small number
of pyramidal cells in CA1 or CA3; 3 = moderate damage to a larger
area in one hippocampal region (CA1 or CA3); 4, severe damage to two
hippocampal regions; 5 = extreme damage or substantial neuronal
death in more than two regions, i.e. CA1, CA3, CA4, or
dentate gyrus. Sections were scored blind and independently by two investigators.
Electrophysiology--
Transverse vibratome sections (400 µm)
were at all times bathed in artificial cerebrospinal fluid,
i.e. 124 mM NaCl, 5 mM KCl, 26 mM NaHCO3, 1.24 mM
KH2PO4, 2.4 mM CaCl2,
1.3 mM MgSO4, and 10 mM glucose,
saturated with 95% O2 and 5% CO2.
Electrophysiological recordings, done at least 3 h after
dissection to allow recovery, were made with bipolar tungsten
microelectrodes to stimulate Schaeffer's collaterals. Evoked field
excitatory postsynaptic potentials (fEPSP) were recorded in CA1 with
low resistance (2 megohm) glass microelectrodes filled with 2 M NaCl. Test stimuli were 0.1-ms constant voltage pulses
delivered every 30 s at an intensity sufficient to evoke an
~33% maximal response. LTP was induced by high frequency stimulation either 2 trains of 1-s pulses of 100 Hz separated by 20 s with each pulse 0.2 ms (strong stimulation), or a single train of 0.4 s, 100 Hz, with each pulse 0.1 ms (weak stimulation). The slope of
fEPSP (mV/ms) was averaged from four consecutive responses. Paired-pulse facilitation was measured by the relative ratio of the
slope of the second to the first fEPSP.
Acute Dissociation of Hippocampal Neurons from Adult and
Newborn Mice--
Neurons from adult mouse hippocampi (32) were
dissociated from sections (200 µm) incubated at 32 °C in
oxygen-saturated buffer (120 mM NaCl, 5 mM KCl,
1 mM CaCl2, 1 mM MgCl2,
20 mM PIPES, 25 mM glucose (pH 7.0) containing
0.8 mg/ml trypsin) for 90 min. Slices were rinsed, triturated in
Dulbecco's minimal essential medium (DMEM) with fire-polished glass
pipettes, and plated on polylysine-coated coverslips. Neurons were
allowed to attach, and the medium was replaced by HEPES-buffered saline
used for dye loading and recording.
Primary hippocampal neurons from newborn mice were plated on astrocyte
monolayers treated with the antimitotic agents 5-flouro-2-deoxyuridine (8.1 mM) and uridine (20.4 mM). Astrocyte
feeder layers were prepared from the spinal cords of newborn mice by
mechanical and enzymatic dissociation with trypsin. Cells were
suspended in DMEM with 10% fetal calf serum, 2 mM
glutamine, and antibiotics. Cells were plated (3 × 103 cells/well) in 4-well cluster plates on coverslips coated with
polylysine and collagen.
Hippocampi from newborn mice were dissociated with papain (25 units/ml)
activated with cysteine (2 mM) in DMEM for 60 min at
37 °C before transfer to DMEM with fetal calf serum (5%), N2 supplement, and antibiotics. Cells were triturated and plated on
astrocyte feeder layers. After attachment (3-6 h after plating), the
medium was changed to neurobasal medium with B27 supplement (Life
Technologies, Inc.). Experiments were performed on neurons cultured for
19-21 days in Locke's buffer, i.e. 154 mM
NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 1.0 mM MgCl2, 3.6 mM
NaHCO3, 5 mM glucose, 5 mM HEPES
(pH 7.2).
Microfluorometric Calcium Measurements--
Cytoplasmatic
calcium was measured by fluorescence ratio imaging with
acetoxymethyl-fura 2 (fura-2/AM). For dye loading, cells on coverslips
were incubated in Locke's buffer with Fura-2/AM (5 µM)
for 30 min at 37 °C, washed, and imaged under an upright microscope.
Average intracellular calcium ion concentration
([Ca2+]i) in individual neurons was calculated
from fluorescence using a digital imaging system (Till Photonics,
Munich, Germany). Measurements were acquired at 1-3-s intervals at
dual excitation wavelengths, and digital fluorescence images were
constructed. [Ca2+]i was determined for each
pixel in the frame with fluorescence intensities over a given threshold
and using the relation [Ca2+]i = Kd[(R Transgenic Mice Expressing Wild-type and Mutant
Presenilins--
Transgenic mice that express either human
wild-type PS1 or PS2 or EOFAD mutants PS1[A246E] or PS2[N141I] were
generated using the mouse thy-1 gene promoter (28) to
assure expression in neurons only (1, 2, 26). For each construct, 2-6
independent founders were generated, and the selection of strains was
based on human transgene mRNA and protein levels in brain (results
not shown). Human PS transgenic proteins were present at levels similar
to endogenous murine PS1 in wild-type mouse brain, indicating that saturating replacement was effective (see Refs. 1 and 2 for references
and discussion). Western blotting demonstrated proteolytic processing
of human PS1 transgenes into N- and C-terminal fragments (1, 2)
(results not shown). For current experiments we used 2 transgenic lines
expressing PS1[A246E], i.e. lines 2 and 4 compared with 2 PS1[wt] transgenic strains 7 and 8. APP metabolism was disturbed in
the brain of double transgenic mice that coexpress mutant PS1[A246E]
and human APP/London (1, 2). Histologically, the brains of all PS
transgenic mice, including PS1[A246E] and PS2[N141I] mice up to 2 years old, were normal by standard hematoxylin-eosin, silver, and
thioflavin-S staining (1, 2) (results not shown). Learning and spatial
memory of PS1[A246E] transgenic mice were unaffected in the water
maze test, in which neither the escape latency nor escape pathway was different from PS1[wt] transgenic mice at 3 and 9 months of age (1,
2) (results not shown).
Excitotoxicity in Response to Kainic Acid--
KA elicited
significant higher seizure activity for the same dose in 3 independent
strains of mutant transgenic mice, i.e. 2 strains of
PS1[A246E] and the PS2[N141I] strain. Wild-type human PS1 or PS2
transgenic mice reacted in a manner similar to nontransgenic mice
(Table I). Neuronal damage 24 h
after KA was rated on a scale of 1 to 5 (specified under
"Experimental Procedures"). In the hippocampus of nontransgenic
mice, confirmed as the area most vulnerable to KA (31), the
average neuron death varied from minor to mild for doses of 12 and 16 mg KA/kg, respectively (Table II).
Neuronal damage was confined largely to single or small clusters of
pyramidal cells in CA3, known to be most susceptible to KA (31, 34).
Damage was always absent from the CA2 and dentate gyrus in all mice,
confirming that these structures are unaffected by KA (35, 36).
Considerably more neuronal damage was evident in mutant PS1 and PS2
transgenic mice. In two different PS1[A246E] strains and in
PS2[N141I] mice, average neuronal damage at each dose of KA was
significantly higher than in nontransgenic or wild-type PS1 or PS2
transgenic mice (Table II; results not shown).
Dantrolene (30) administered prior to KA in PS2[N141I] transgenic
mice significantly reduced or even completely prevented neuronal damage
from KA, with only a minimal lowering of seizure activity (Table
III). In a smaller scale experiment,
mutant PS1 transgenic mice were also protected against KA by
dantrolene (results not shown).
Hippocampal LTP--
Hippocampal LTP was measured at synapses
between Schaeffer's collaterals and CA1 pyramidal neurons (26, 37,
38). LTP evoked by strong tetanic stimulation was not different between PS1 transgenic mice (Fig. 1A).
In contrast, weaker stimulation, i.e. 1 train of 0.4 s
at 100 Hz with 0.1-ms pulses, induced stronger LTP in the hippocampus
of PS1[A246E] transgenic as opposed to PS1[wt] transgenic mice or
nontransgenic mice. After 2 h the fEPSP slope in PS1[A246E]
transgenic mice was 169.2 ± 30.3% as opposed to 119.9 ± 18.2% in PS1[wt] transgenic mice (p < 0.05) (Fig.
1B).
Paired-pulse facilitation, a reflection of the amount of neuromediator
ejected because of the residual increase in
[Ca2+]i in presynaptic terminals after the first
pulse, was measured using interpulse intervals ranging from 25 to 100 ms. Independent of the interval, paired-pulse facilitation was similar in all mice. At 50-ms interpulse intervals, the ratio (second to first
stimulus) of slopes of fEPSP was 168.7 ± 36.1% in nontransgenic mice, 163.7 ± 22.9% in PS1[A246E] transgenic mice, and
165.5 ± 20.2% in PS1[wt] transgenic mice.
[Ca2+]i in Acutely Dissociated Neurons of
Adult Mice--
No significant differences were observed in basal
[Ca2+]i in the neurons of adult and aged
PS1[wt] and PS1[A246E] mice. Potassium ions triggered a rapid
increase in [Ca2+]i in the soma of adult
PS1[wt] and PS1[A246E] neurons. Peak values (1-3 s) were
significantly higher in PS1[A246E] neurons (Fig.
2), whereas in neurons derived acutely
from old transgenic mice even higher values were measured. The
normalization of medium potassium ion levels led to a progressive
decline of [Ca2+]i to initial resting levels
(Fig. 2). Glutamate (50 µM) increased peak
[Ca2+]i significantly more in PS1[A246E]
relative to PS1[wt] neurons (Fig. 3).
Again, in neurons from old PS1[A246E] transgenic mice, the peak
[Ca2+]i induced by glutamate was significantly
higher than in young PS1[A246E] mice (Fig. 2). PS1[A246E]
hippocampal neurons, in parallel with PS1[wt] neurons exposed to
increasing concentrations of glutamate, were much more sensitive to
glutamate-triggered excitotoxicity (Fig. 3). This finding corroborated
our own in vivo observations (see above) and reported
results on hippocampal neurons from unrelated PS1[M146V] transgenic
mice (12).
[Ca2+]i in Cultured Neurons--
In
cultured hippocampal neurons from newborn mice, basal
[Ca2+]i was similar independent of the mouse
genotype (Fig. 4). Potassium ions caused
a rapid [Ca2+]i increase in all cultures with a
similar time course; i.e. similar initial peak after depolarization
followed by a rapid decline to a plateau that was somewhat lower in
PS1[A246E] neurons (Fig. 4). The wash-out of potassium ions
progressively restored [Ca2+]i to initial resting
levels in all cultures of nontransgenic, PS1[wt] and PS1[A246E]
neurons (Fig. 4).
Again, glutamate induced a rise in [Ca2+]i that
was significantly higher in neurons from PS1[A246E] mice (Fig. 4)
than in acutely dissociated adult neurons (Fig. 2). The addition
of NMDA (70 µM with 10 µM glycine)
resulted in comparable responses in PS1[wt] and PS1[A246E]
hippocampal neurons (Fig. 4C) indicating that NMDA receptors
did not contributed differentially.
Bradykinin (1 µM) increased [Ca2+]i
significantly more in PS1[A246E] neurons than in nontransgenic and
PS1[wt] transgenic neurons (Fig. 4D). This extends the
findings in fibroblasts from PS1[M146V] transgenic mice (24) to the
most relevant cell type and demonstrates that these responses are
mediated by metabotropic glutamate receptors. IP3 receptor
protein levels were not different in the brains of wild-type and
transgenic mice on Western blots (data not shown).
The depletion of the ER stores with thapsigargin (1 µM)
significantly increased [Ca2+]i in PS1[A246E]
hippocampal neurons relative to PS1[wt] neurons (Fig. 4E).
Collectively, the data indicate that the homeostasis of intracellular
Ca2+ was severely disturbed by mutant presenilins by a
mechanism(s) that involves the dynamics of ER calcium stores.
Overexpression of EOFAD mutants PS1[A246E] or PS2[N141I]
in the brain of transgenic mice resulted in no major pathological manifestations, even in old mice, as observed by us and others (1, 2,
12, 39). This is remarkably different from humans, because mutant PS1
causes the most aggressive cases of EOFAD (40). On the other hand, in
combination with human APP, PS1[A246E] was extremely pathogenic,
i.e. it increased amyloid in brain parenchym and
cerebral blood vessels (Refs. 1 and 2 and references therein). The
proposed relationship of PS1 to Mice expressing mutant PS1[A246E] or PS2[N141I] were more sensitive
to kainic acid as evident from the acute seizure intensity and delayed
neuronal damage. The neurotoxicity of KA obligatory involves glutamate
receptors and results in cellular Ca2+ overload, confirmed
by direct measurements here and in mice with a different PS1 mutant
(12). Dantrolene, an effective inhibitor of calcium release from the
ER, effectively protected mutant PS transgenic mice from
excitotoxicity. This finding supports experimentally the notion that
mutant PS contribute to neurotoxicity by disturbing calcium ion
homeostasis in and from the ER.
This conclusion joined seamlessly with our findings of facilitated
induction of LTP and with related aspects of disturbed calcium
homeostasis, which were published recently during the course of this
work (12, 20-24). Classic LTP was not different in PS transgenic mice
when induced by a strong stimulus, in contrast to weak stimulation that
elicited LTP only in mutant PS1 mice. Clearly, mutant PS1 decreased the
threshold for LTP without affecting its maximum amplitude. The
induction of LTP in CA1 operates through Ca2+ influx via
NMDA receptors and L-type voltage-gated calcium channels (37,
41). Release from the ER is debated and largely based on depletion of
internal stores by thapsigargin, which blocks LTP induced by weak but
not strong tetanization (42-44). The release of Ca2+ from
the ER is mediated by IP3 and ryanodine receptors (RyR). Blocking metabotropic glutamate receptors prevents both the generation of IP3 and the induction of LTP (45). The effect of
bradykinin supports an IP3-mediated effect of PS mutants,
which are anchored in the ER and could directly effect Ca2+
release by IP3 receptors. LTP is triggered evidently by
NMDA receptors, but the reaction of [Ca2+]i to
NMDA was unaffected in mutant PS1 neurons. Although RyR expression
might be disturbed by mutant PS1 (46), the potassium ion depolarization
results in cultured mutant PS1 neurons argue against involvement of RyR
in our experimental system.
Therefore, the detailed mechanism of [Ca2+]i
increase following glutamate in neurons of PS1[A246E] mice remains to be defined. The significant response of PS1[A246E] neurons (present work), of fibroblasts from PS1[M146V] mice (24), and of PS1 mutant
PC12 cells to thapsigargin (47) all suggest an increased pool of
Ca2+ ions available for release. Although this
points to the ER and is most consistent with the localization of PS in
the ER, the complex cellular responses to calcium influx make it as yet
impossible to identify the primary effect of mutant PS on the dynamic
regulation of [Ca2+]i, referring also to
capacitive calcium entry (48) or RyR expression (46).
Finally, and most intimately related to AD pathology, we must address
the apparent contradiction of mutant PS affecting calcium homeostasis
and PS in or as
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), especially A
42 (4).
Presenilins (PS) contain 6 to 8 transmembrane domains and are
predominantly confined to the endoplasmic reticulum (ER). Similar to
Caenorhabditis elegans sel-12 and
spe-4, PS might function in signal transmission and
intracellular transport (5, 6). More recently PS1 was claimed to be
-secretase (7), the elusive proteinase responsible for cleaving A
from APP (8).
, the proposed primary defect in EOFAD,
although perturbation of intracellular fluxes of calcium ions could be
the main problem in sporadic AD (9). Whether this causes or follows
from increased A
, by affecting APP processing and/or clearance, is
disputed (10-14). Others took failing calcium homeostasis even further
to include apoE4 (15, 16), while even more provocative is the
"reversed amyloid hypothesis" in which A
and neurofibrillary
tangles are "executers" following disrupted calcium homeostasis
(17).
, resulting
in more amyloid plaques earlier in life (4, 18, 19) and aggravating
cerebrovascular angiopathy (2) by a mechanism different from aging (1).
Recently, enhanced long-term potentiation (LTP) or facilitated synaptic
transmission was observed and related to altered calcium homeostasis in
brain slices of mutant PS1 transgenic mice (12, 20-24). This finding enforced our ongoing analysis of PS1 and PS2 transgenic mice, focusing on glutamate-mediated neurotransmission, thought to be compromised in AD patients (25).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Rmin)/(Rmax
R)](F0/Fs) (33).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Seizures
Neuronal damage
Dantrolene
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Fig. 1.
Hippocampal LTP induced by strong and weak
stimulation. A, relative changes in fEPSP slopes as a
function of time after strong high frequency stimulation,
i.e. two trains of 1 s with pulses of 0.2 ms at 100 Hz.
All points are the mean ± S.E. of 6 experiments on sections from
6 distinct mice with the indicated genotype. B, relative
changes in fEPSP slopes as a function of time after weak HFS
stimulation, i.e. trains of 0.4 s at 100 Hz with 0.1-ms
pulses. Inset, examples of typical fEPSP recorded 2 h
after HFS (traces b and d) superimposed on
control fEPSP (traces a and c) for PS1[A246E]
transgenic mice (left) and PS1[wt] transgenic mice
(right).
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Fig. 2.
Effects of potassium depolarization and
glutamate on [Ca2+]i in acutely dissociated
neurons. A, typical changes in
[Ca2+]i in individual cells of acutely
dissociated hippocampal neurons from adult PS1[wt] and PS1[A246E]
mice (4-6 month) and old PS1[A246E] mice (14-16 month). Cells were
treated subsequently by 50 mM K+ and 50 µM glutamate (indicated by lower bars).
B, values of the mean (m) and peak (p)
values from 10-15 cells analyzed in four individual mice obtained as
indicated in panel A (top). Statistical
significance *, p < 0,01; **, p < 0,001.
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Fig. 3.
Effect of glutamate concentration on neuronal
survival. Hippocampal neurons from PS1[wt] (open
squares) and PS1[A246E] transgenic mice (closed
squares) exposed for 24 h to glutamate. Survival was
quantified by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay. Values are the mean ± S.E. of 3 cultures.
Statistical significance: * p < 0,01, * *,
p < 0,001.
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Fig. 4.
Effect of potassium depolarization and
glutamate on [Ca2+]i in primary hippocampal
neurons. A, typical changes in
[Ca2+]i in individual cell somata of primary
cultured hippocampal neurons from newborn nontransgenic mice
(FVB) and from PS1[wt] and PS1[A246E] transgenic mice.
See Fig. 3 for experimental details. B, mean (m)
and peak (p) values from cells (n = 20-30)
analyzed at the time intervals indicated in panel A.
Statistical significance: *, p < 0,01; **,
p < 0,001. C,
[Ca2+]i basal and following the addition
of NMDA (70 µM NMDA with 10 µM glycine) in
at least 20 cells from 4 cultures (mean ± S.E.). D and
E, peak [Ca2+]i in response to
bradykinin (BK, 1 µM) (D) and
thapsigargin (TG, 1 µM) (E) in
hippocampal neurons of nontransgenic (FVB) and PS1[wt] and
PS1[A246E] transgenic mice. The mean ± S.E. of the rise in
[Ca2+]i in at least 20 cells in 4 independent
cultures is shown. Statistical significance: **, p < 0,001.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-secretase (7, 8) should not
distract from the fact that the exact gain-of-function of mutant
presenilins in vivo is unknown. Therefore, PS1[A246E] and
PS2[N141I] transgenic mice were analyzed for (i) reactivity to kainic
acid, (ii) hippocampal LTP, and (iii) regulation of [Ca2+]i. In all three paradigms,
glutamate-mediated neurotransmission is implicated as in the
pathophysiology of AD by the concept of excitotoxicity (25).
-secretase activity (7, 8). It is conceivable
that all of the effects on calcium homeostasis of mutant PS are
indirect and are mediated by increased cellular levels of amyloid
peptides. None of the available systems allows one to accept or dismiss
this possibility, whereas on the contrary, very recent evidence
strongly supports the opposite view (48). To clarify this
hypothesis, we have generated additional mouse strains that
express mutant PS1 in an APP-deficient background, in addition to mice
that express mutant APP[V717I] in a conditional, neuron-specific
PS1-deficient background. These multiple transgenic mouse strains are
now being characterized to define whether and how mutant PS1 affects
neuronal calcium homeostasis in the absence of amyloid peptides.
Increased [Ca2+]i in cultured cells increased
A
production (11) while also increasing hyperphosphorylation of
protein tau (49). This link to both major neuropathological lesions in
AD makes the deregulation of intracellular calcium homeostasis a prime
candidate for all AD cases, both familial and sporadic.
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ACKNOWLEDGEMENTS |
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The contributions of many scientists are gratefully acknowledged, particularly those of P. St. George-Hyslop, P. Seubert, and H. Van der Putten.
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FOOTNOTES |
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* This investigation was supported by Deutsche Forschungs Gemeinschaft (DFG SFB406), Fonds Wetenschappelijk Onderzoek-Vlaanderen, National Fonds voor Wetenschappelijk Onderzoek (NFWO)-Lotto, Queen Elisabeth Fund for Medical Research, Inter-university Poles of Attraction Program of the Federal Office for Scientific Affairs (Belgium), Flemish Action Program for Biotechnology (IWT/VLAB, COT-008), 4th and 5th Framework European Economic Commission programs, the Rooms Fund, and KU Leuven R&D.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.
** Post-doctoral fellow of the KU Leuven research fund.
Research assistant of the Belgian National Fund for Scientific Research.
¶¶ To whom correspondence should be addressed: Experimental Genetics Group, Center for Human Genetics, Campus Gasthuisberg O&N 06, B-3000 Leuven, Belgium. Tel.: +32-16-3458-62; Fax: +32-16-3458 71; E-mail: fredvl@med.kuleuven.ac.be.
Published, JBC Papers in Press, January 23, 2001, DOI 10.1074/jbc.M010977200
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ABBREVIATIONS |
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The abbreviations used are:
AD, Alzheimer's
disease;
PS, presenilin(s);
EOFAD, dominant early onset familial
Alzheimer's disease;
APP, amyloid precursor protein;
ER, endoplasmic
reticulum;
A, amyloid peptide;
LTP, long-term potentiation;
KA, kainic acid;
fEPSP, excitatory postsynaptic potentials;
PIPES, 1,4-piperazinediethanesulfonic acid;
DMEM, Dulbecco's minimal
essential medium;
NMDA, N-methyl-D-aspartic
acid;
IP3, inositol 1,4,5-trisphosphate;
RyR, ryanodine
receptor.
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