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INTRODUCTION |
Alzheimer's disease
(AD)1 is identified by a
progressive decline in cognitive functions and is characterized by
neuropathologies including senile plaques comprising
-amyloid (A
)
peptide, neurofibrillary tangles, and ultimately neuron loss. Mutations
in the A
precursor protein (APP) and in the presenilin
(PS1 and -2) genes have been identified, which
co-segregate with familial Alzheimer's disease (FAD) (1, 2).
Overexpression of the mutated genes in cells and in transgenic animals
has been shown to mimic some of the features of AD, including increased
accumulation of A
(1, 3-5). Most significantly, transgenic mice
also show impairments in neuronal function (5) and develop deficits in
learning and memory (4), although neuron loss is not a consistent
feature of the phenotype (4-7). Although it is known that A
is
neurotoxic in vivo and in vitro (8), there are
indications that cognitive impairment may precede both high levels of
A
accumulation and pronounced neuronal degeneration in the brain (5,
9-11). Because of the protractive and progressive nature of the
disease, A
may be present in the brain at sublethal concentrations
for extended periods. Although at these levels A
does not compromise
neuron survival, it may affect critical signal transduction processes that mediate plastic neuronal changes, including those involved in
learning and memory. The transcription factor CREB, which regulates expression of cAMP response element (CRE)-containing genes, plays an
essential role in learning and memory processes in a variety of species
ranging from Drosophila to mammals (12-15). Phosphorylation at Ser-133 is critical for the transcriptional activity of CREB (16-18). Neuronal activity-dependent phosphorylation of
Ser-133 of CREB has been well documented, and in cultured neurons, both NMDA receptor activation and membrane depolarization can lead to the
activation of CREB (17-19). Disruption of CREB function specifically
interferes with activity-dependent synaptic plasticity ranging from long term potentiation (LTP) to long term memory (13-15).
It is expected, therefore, that mechanisms that interfere with CREB
activation would compromise CREB activity-dependent neuronal function through disruption of downstream gene expression.
Brain-derived neurotrophic factor (BDNF) is one of the target
genes of CREB (17, 18). BDNF, a member of the neurotrophin family,
enhances survival, differentiation, and growth of certain neuronal
populations, modulates synaptic activity, and acts as an effector of
neuronal plasticity both during development and in the adult (20, 21).
BDNF participates in LTP, is up-regulated in the hippocampus during
learning (22), and deficits in BDNF compromise LTP and learning and
memory (20). BDNF mRNA and protein are reduced in the hippocampus
in AD (23-25), a reduction proposed to contribute to cognitive decline
observed in AD. Thus, examination of BDNF transcription provides a
means of assessment of effects on CREB regulation, which may play a
significant role in the pathogenesis of AD.
Here, we report that levels of A
-(1-42), which do not affect the
survival of cortical neurons, may indeed interfere with functions
critical for neuronal plasticity, by eliciting a reduction of the
activity-dependent phosphorylation of CREB and the
expression of BDNF, one of the important target genes of this
transcription factor.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Cultures greatly enriched in cortical neurons
from embryonic day 18 rat fetuses were prepared as described previously
(26). Cells plated at 2.5 × 104 cells/cm2
were cultured in poly-L-lysine-treated multiwell plates and
maintained in serum-free optimal Dulbecco's modified Eagle's medium
supplemented with B-27 components (Life Technologies). When cells were
exposed to A
, the medium was switched to Dulbecco's modified
Eagle's medium/B27 containing A
. Cultures were maintained for 5 days before treatments. Neuronal survival was assessed by trypan blue exclusion (26) and in select experiments using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay (27) and annexin V binding to externalized phosphatidylserine to monitor early changes in putative apoptotic processes (28); these methods gave comparable results.
Tetrodotoxin (1 µM) was added 2 h before
treatments to reduce endogenous synaptic activity. Cells were
stimulated using either 30 mM KCl (high K+) for
15 min or 10 µM NMDA for 10 min. Amino-5-phosphonovaleric acid (100 µM) was added to the cultures 30 min before the
high K+ exposure.
A
Preparation--
A
peptides were synthesized by
solid-phase N-(9-fluorenyl)methoxycarbonyl (Fmoc) amino acid
chemistry, purified by reverse-phase high performance liquid
chromatography, and characterized by electrospray mass spectrometry as
described previously (26, 29). A stock solution of A
-(1-42) (1 mM) was prepared in distilled water and used after one
freeze-thaw cycle. We have documented previously that A
-(1-42)
peptides prepared this way assemble into
-sheet fibrils as
determined by thioflavin staining, circular dichroism, electron
microscopy, and sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and are toxic to nerve cells (26, 29, 30). To
facilitate comparisons, preparations of A
-derived diffusible ligands
(ADDL) were obtained from the same lot of A
-(1-42) peptides as the
-sheet containing A
-(1-42) preparations described above. Solutions of ADDLs were prepared as described previously (31, 32). In
brief, A
-(1-42)-containing solutions (0.34 mg/ml) were incubated
with clusterin (0.17 mg/ml) at 22 °C for 24 h. Solutions were
centrifuged at 14,000 × g for 10 min to remove large
aggregates, and the supernatant was used for all assays. In agreement
with the published features of these preparations, microscopic
examination indicated that the supernatant contained no large aggregates.
Western Blotting--
Cortical neurons were lysed in SDS sample
buffer, and the proteins were resolved by SDS- polyacrylamide gel
electrophoresis (10% acrylamide) and transferred to polyvinylidene
difluoride membrane. Membranes were incubated at room temperature in
PBS containing 5% nonfat milk for 60 min to block the nonspecific binding. Following incubation with the primary antibodies specific for
either Ser-133 phosphorylated CREB (P-CREB) (1:2,000) or total CREB
(T-CREB), which recognize both phospho- and dephospho-CREB (1:1,000)
(both antibodies from Upstate Biotechnology, Inc.), the blots were
washed in PBS containing 0.1% Tween and then incubated with the
secondary antibody, goat anti-rabbit IgG conjugated with horseradish
peroxidase (Vector Laboratories), at 1:8000 dilution in the blocking
solution for 60 min. Blots were then washed four times with PBS
containing 0.1% Tween. Immunolabeling was detected by enhanced
chemiluminescence (ECL, Amersham Pharmacia Biotech) according to the
recommended conditions. Immunoreactivity was quantified using
densitometric analysis.
Immunocytochemistry--
Cultures were initially treated with a
solution of 3% aqueous H2O2 for 3 min, then
briefly rinsed in Tris-buffered saline (TBS, 0.05 M Tris in
0.9% NaCl, pH 7.4), and incubated in TBS containing 5% normal goat
serum. The cultures were then incubated at 4 °C in TBS, 5% serum
containing the rabbit polyclonal IgG against P-CREB (1:2000). Cultures
were then rinsed in buffer and incubated in biotinylated goat
anti-rabbit IgG. After a buffer rinse, cultures were incubated in the
presence of avidin-biotin-horseradish peroxidase complex (Vector
Laboratories) for 90 min. They were then rinsed in 0.1 M
Tris-HCl, pH 7.6, and incubated in Tris-HCl containing 0.05%
diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide for
5-10 min. Reactions were stopped by rinsing the cultures with PBS.
Quantitative analysis was performed by Image Pro-Plus software (Media Cybernetics).
Cortical Neuron Transfection and Luciferase Assay--
Cortical
neurons were transfected with plasmid pIII(170)Luc (a kind gift of Dr.
Michael E. Greenberg, Harvard Medical School) at 3 DIV using a
procedure described previously (33). Briefly, all transfections were
conducted in 6-well 35-mm dishes with LipofectAMINE (Life Technologies,
Inc.) according to the manufacturer's instructions. One well was
transfected with 1 µg of reporter plasmid and 0.1 µg of pRL-CMV
(Promega), a cytomegalovirus-luciferase control plasmid to normalize
BDNF exon III promoter activity. Forty hours after transfection,
cultures received 30 mM KCl for 9 h and then plates
were washed twice with cold PBS, and cells were lysed with 200 µl of
lysis buffer (Promega). Twenty µl of cell extract were used for a
dual-luciferase reporter assay (Promega) according to the
manufacturer's instructions.
RT-PCR--
BDNF exon III and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) mRNA contents were estimated using RT-PCR.
Total RNA was isolated from cultures at 5 DIV by a spin column kit
(Promega), and RT-PCR was carried out using a one-tube RT-PCR
kit (Amersham Pharmacia) following the manufacturer's instructions.
100 ng of total RNA was used, and the single-stranded cDNA was
amplified by PCR with exon-specific primers. The primers for BDNF exon
III and GAPDH were described previously (18). BDNF exon III primers were as follows: reverse primer E5R, 5'-GAGAAGAGTGATGACCATCCT-3' and
forward primer E3F (exon III-specific), 5'-TGCGAGTATTACCTCCGCCAT-3'. GAPDH primers were 5'-TCCATGACAACTTTGGCATCGTGG-3' and
5'-GTTGCTGTTGAAGTCACAGGAGAC-3'. The amount of total RNA in the samples
was normalized to the amount of GAPDH. The PCR products were separated
by electrophoresis on 6% polyacrylamide gels and stained using Vistra
Green (Amersham Pharmacia Biotech). The products were quantified by
PhosphorImager (Molecular Dynamics) analysis.
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RESULTS |
Low Concentrations of A
-(1-42) Decrease Neuronal
Activity-dependent Phosphorylation of
CREB--
-Amyloid peptides, such as A
-(1-42) and
A
-(25-35), are known to compromise neuronal survival both in
vitro and in vivo (8, 34). The effect of A
-(1-42)
on cultured neurons is concentration-dependent, and
significant cell loss was detected after treatment at concentrations of
>20 µM. We also observed that under our experimental
conditions there was a threshold for the effect of A
-(1-42) on cell
viability. Twenty four-hour exposure of cortical neurons to
A
-(1-42) at
20 µM with the lot of peptide used in
this study resulted in significant cell loss, whereas survival was not
affected at
10 µM (not shown). A concentration range of
1-10 µM was therefore chosen for the exploration of the
influence of A
-(1-42) on neuronal function. It should be noted that
no studies to date have assessed the effect of sublethal levels of A
on the regulation of gene expression that is mediated by
activity-dependent signal transduction.
Cognitive dysfunction is a characteristic feature of the AD phenotype.
Recent studies have demonstrated that synaptic plasticity, including
certain forms of LTP and memory, depends critically on the activation
of the transcription factor CREB (13-15). Therefore, we examined the
influence of sublethal concentrations of A
-(1-42) on
activity-dependent CREB phosphorylation.
K+-induced depolarization of neurons is known to elicit the
phosphorylation of CREB at Ser-133, an event that is essential for its
transcription-activating function (16-18). Therefore, the level of
activated CREB was examined with Western blotting using a specific
antibody against Ser-133-phosphorylated CREB (P-CREB). Pretreatment
with 5 or 10 µM A
-(1-42) for 1 h decreased high
K+-induced elevation of the amount of P-CREB (Fig.
1, A and B). Pretreatment with 5 or 10 µM A
-(1-42) for 1 h on
unstimulated cells had no significant effect on the basal level of
P-CREB (as a percentage of control the values after exposure to 5 or 10 µM A
-(1-42) 115 ± 33 and 120 ± 21%)
(Fig. 1C).

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Fig. 1.
Treatment with
A -(1-42) decreased high
K+-induced CREB phosphorylation in cultured cortical
neurons. CREB activation was examined using an antibody against
Ser-133-phosphorylated CREB (P-CREB). A, Western blot
analysis of CREB. In comparison with control, the amount of P-CREB
increased by exposing cortical neuronal cultures to 30 mM
KCl for 15 min. Pretreatment with sublethal concentrations of
A -(1-42) (5 or 10 µM) for 1 h resulted in a
decrease in the amount of P-CREB. B, quantification of
levels of P-CREB and T-CREB obtained in three independent experiments
shows that A -(1-42) treatment resulted in a
concentration-dependent suppression of the
K+-induced elevation of P-CREB content (*,
p < 0.05) but had no significant effect on the level
of total CREB (C, control; K, 30 mM
KCl; A1, A5, and A10,- 30 mM KCl
after pretreatment with 1, 5, and 10 µM A -(1-42),
respectively). C, pretreatment with 5 or 10 µM
A -(1-42) for 1 h on unstimulated cells had no significant
effect on the basal level of P-CREB. D, pretreatment with 5 µM A -(1-42) for 1 and 24 h decreased the amount
of P-CREB in high K+-treated cells. E,
pretreatment with 10 µM A -(1-42) with random sequence
A (R) had no significant influence on the high K+-induced
increase of P-CREB levels. Similar results were obtained in three
independent experiments.
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The main effect of A
-(1-42) on the depolarization-induced elevation
of P-CREB occurred within the 1st h, and in cultures exposed to high
K+ for 15 min the P-CREB level was, as a percentage of that
in the untreated controls, 50 ± 3.0 or 40 ± 2.0% after
treatment with 5 µM A
-(1-42) for 1 or 24 h (Fig.
1D; n = 3). In contrast, the effect of
A
-(1-42) with random sequence (A
-(1-42)(R)) failed to influence
the high K+-induced increase of P-CREB levels (Fig.
1E). After a 1-h treatment with 10 µM
A
-(1-42)(R) the estimates were, as a percentage of those in the
untreated controls, 91 ± 6.7% compared with less than 40%
observed after exposure to 10 µM A
-(1-42) (Fig.
1B). None of the treatments had significant effects on total
CREB levels (e.g. Fig. 1B).
We examined further CREB activation using immunocytochemistry and
observed that membrane depolarization induced by exposure to elevated
K+ resulted in almost all cells in a pronounced increase in
P-CREB immunoreactivity, which was markedly attenuated by treatment
with A
-(1-42) (Fig. 2).

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Fig. 2.
Immunohistochemical analysis of
CREB phosphorylated at Ser-133. A, P-CREB immunoreactivity
increased in cortical neuronal cultures exposed to 30 mM
KCl as compared with controls. Treatment with 5 µM
A -(1-42) for 1 h suppressed the high K+-induced
increase in P-CREB content. B, quantification of P-CREB
immunoreactivity. C, control; K, cultures treated
with 30 mM KCl; K+A, high K+-treated
cultures exposed to 5 µM A -(1-42). Estimates
represent the mean ± S.E. of three independent experiments.
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Diffusible A
-(1-42) Oligomers Decrease Neuronal
Activity-induced Phosphorylation of CREB--
There is evidence that
neurotoxicity in primary cultures is related to the ability of
A
-(1-42) to form fibrillary assemblies (3, 8, 26). In addition,
A
may form small diffusible oligomers (referred to as ADDLs, for
A
-derived diffusible ligands), which are highly neurotoxic (31, 32).
In the ADDL preparation used in the present study the formation of
large A
assemblies was reduced using clusterin, and the large
assemblies that did form were removed by centrifugation,
according to the procedure of Lambert et al. (32) (see
"Experimental Procedures"). Most importantly, ADDLs cause
degeneration of neurons at lower concentrations than conventionally
assembled preparations (32) and may share properties with soluble A
that is present in the AD brain. Also under our experimental
conditions, ADDLs compromised neuronal survival at concentrations lower
than those needed to effect neurotoxicity with conventional fibrillar
A
-(1-42) preparations, causing significant cell loss (23%),
already at a concentration of 1 µM after 24 h of
exposure. Furthermore, at the sublethal concentrations of 100 nM, ADDL elicited a marked suppression of the high
K+-induced increase in P-CREB content to 62 ± 13.6%
(n = 3) of the levels in the untreated controls (Fig.
3).

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Fig. 3.
Treatment with ADDL
(A -(1-42)-derived diffusible ligand)
decreased high K+-induced CREB phosphorylation in cultured
cortical neurons. Western blot analysis of P-CREB: pretreatment
with 100 nM ADDL for 1 h attenuated the elevation of
P-CREB content induced by exposure of cortical neurons to 30 mM KCl for 15 min.
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To determine whether the observed effects were specific to
A
-(1-42), the influence of A
-(25-35), a toxic fragment of A
, was examined. This fragment also elicited neuron loss at
25
µM but, importantly, did not interfere with CREB
phosphorylation at the sublethal concentration of 10 µM
(Fig. 4, A and B).
Therefore, both the fibrillar A
-(1-42) and the diffusible
A
-(1-42) oligomers (ADDLs) interfere at sublethal concentrations
with neuronal activity-induced signal transduction via CREB, whereas
the potent toxic fragment A
-(25-35) is inactive.

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Fig. 4.
Treatment with
A -(1-42) but not
A -(25-35), decreased high
K+-induced CREB phosphorylation in cultured cortical
neurons. A, Western blot analysis of P-CREB. In
contrast to the marked suppression of the K+-induced
increase of P-CREB levels by 5 µM A -(1-42), 1-h
pretreatment with A -(25-35) at the sublethal concentration of 10 µM had no significant effect. B,
quantification of the effect of A -(25-35) (A25)
versus A -(1-42) (A42) on the
K+-induced increase of the level of P-CREB showed that
significant suppression occurred after pretreatment with A -(1-42)
(*, p < 0.05), but not with A -(25-35)
(n = 3).
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Sublethal Concentrations of A
-(1-42) Decrease NMDA
Receptor-mediated CREB Activation--
CREB phosphorylation can also
be elicited by nerve cell activation through the stimulation of NMDA
receptors that play an important role in neuronal plasticity, including
certain types of LTP (35, 36). A short exposure to NMDA in our cultures also evoked a marked increase in P-CREB levels, and this effect was
suppressed by sublethal concentrations of A
-(1-42) (Fig. 5). As a percentage of P-CREB levels in
the NMDA-treated cultures, pretreatment with 5 µM
A
-(1-42) reduced P-CREB levels to 59 ± 7.2%
(n = 3). None of the treatments had significant effects
on total CREB levels (Fig. 5).

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Fig. 5.
Treatment with
A -(1-42) decreased NMDA-induced CREB
phosphorylation in cultured cortical neurons. A,
Western blot analysis of P-CREB. Pretreatment with 5 µM
A -(1-42) for 1 h attenuated the increase in P-CREB content
evoked by exposure to 10 µM NMDA for 10 min. Similar
results were obtained in three independent experiments. B,
quantification of the effect of pretreatment with 5 µM
A -(1-42) for 1 h; estimates are expressed in terms of P-CREB
levels obtained in the NMDA-exposed cultures and they are mean ± S.E. from three independent experiments. The effect of A -(1-42) was
significant (*, p < 0.05).
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Caspase Activation Does Not Contribute to the A
-(1-42)-mediated
Decrease in the Activity-induced Phosphorylation of CREB--
Although
neurons remained viable in the presence of 1-10 µM
A
-(1-42) during the experimental period, sublethal A
-(1-42) may
have triggered early apoptotic events that affect CREB signaling. Previous studies suggested that caspase activation plays an important role in A
-(1-42)-induced apoptosis in neurons (37, 38). We therefore blocked caspase activities, using the general inhibitor Z-VAD-fmk (39). The cultures in the presence and absence of A
-(1-42) were exposed to Z-VAD-fmk under the conditions (150 µM, 1 h of preincubation) when this compound is
known to elicit effective caspase inhibition in cortical neurons and
PC12 cells (40, 41). The caspase inhibitor had no significant effect on
either the basal or K+-induced phosphorylation of CREB and
did not influence the A
-(1-42)-induced suppression of high
K+-activated CREB phosphorylation (Fig.
6). Thus sublethal A
-(1-42) suppresses CREB signaling through a mechanism(s) independent of caspase
activation.

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Fig. 6.
Z-VAD-fmk did not influence the effect of
A -(1-42) in reducing the high
K+-induced elevation of the P-CREB content. Cultured
cortical neurons at 5 DIV were exposed to 10 µM
A -(1-42) in the presence or absence of 150 µM
Z-VAD-fmk for 1 h, before treatment with 30 mM KCl for
15 min. The experiment was repeated with a similar outcome; Z-VAD-fmk
could not prevent the A -(1-42)-induced suppression of the neuronal
activity-evoked elevation of P-CREB levels.
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Low Concentrations of A
-(1-42) Decrease CRE Transcriptional
Activity and the Induction of BDNF Exon III--
To examine the
consequences of the action of sublethal concentrations of A
on
CREB-mediated signal transduction, we analyzed the expression of one of
the target genes of CREB, BDNF, which plays a critical role in neuronal
survival, differentiation, and plasticity (20). Previous studies showed
that the BDNF gene gives rise to four primary transcripts through
alternative splicing (42) and that the exon III containing mRNA is
the most responsive to neuronal activity in the cerebral cortex and the
hippocampus (17, 18). The effect is mediated largely by
Ca2+ influx, and the promoter of exon III in the BDNF gene
contains a CREB-response element (CRE) (17, 18). As an example of an important CREB-regulated gene, we examined the modulation by
A
-(1-42) of the activity-dependent expression of exon
III-containing BDNF (exon III BDNF). The activation of BDNF exon III
promoter was monitored using a transient transcription activity assay
with luciferase as a reporter gene. Fig.
7A shows that
K+-induced membrane depolarization increased exon III BDNF
promoter activity 3-fold and A
-(1-42) treatment ameliorated the
induction by 31%. We further examined the effect of A
-(1-42)
treatment on the expression of exon III BDNF, employing a quantitative
RT-PCR as described previously (18). Fig. 7, B and
C, shows that exposure of cells to high K+
increased the expression of exon III BDNF by over 3-fold.
Treatment with A
-(1-42) had no effect on the basal amount of mRNA
(not shown) but decreased the membrane depolarization-elicited
elevation of exon III BDNF expression to 38% of the control level.

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Fig. 7.
Effect of A -(1-42)
on BDNF exon III expression in cultured cortical neurons.
A, activity of the BDNF exon III promoter measured by the
luciferase assay. Cortical neurons at 3 DIV were transfected with
plasmid pIII(170)Luc containing the promoter of BDNF exon III
comprising the CRE-like sequence and exon III fused to a luciferase
reporter gene. After 40 h, cultures were switched to fresh medium
and incubated for 1 h in the presence or the absence of 5 µM A -(1-42), before the addition of a solution of KCl
(final concentration 30 mM) or vehicle and further
incubation for 9 h. Transcription activity was assayed by
measuring luciferase activity and expressed as the ratio of the
luciferase activity in extracts from cells exposed to high
K+ to the luciferase activity in extracts from unstimulated
cell. High K+ elicited the activation of BDNF exon III
promoter (K). A -(1-42) treatment suppressed the high
K+-induced activation of BDNF exon III promoter
(K+A). Each value represents the mean ± S.D. of two
independent experiments. B, the expression of BDNF exon III
gene and GAPDH gene (for normalization of the total RNA in the samples)
was measured by RT-PCR. Total RNA (100 ng) was reverse-transcribed into
single-stranded cDNA, and the cDNA was amplified by PCR with
exon-specific primers. The PCR products were separated by
electrophoresis on 6% polyacrylamide gels. C,
quantification of RT-PCR products by PhosphorImager. The amount of
total RNA in the samples was normalized to the amount of GAPDH. Each
value represents the mean ± S.E. from three independent
experiments. Exon III BDNF mRNA levels were significantly increased
in the high K+-exposed cells; pretreatment with 5 µM A -(1-42) significantly attenuated the high
K+-induced elevation of exon III BDNF mRNA levels
(p < 0.05).
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DISCUSSION |
These observations provide the first evidence that sublethal
levels of A
-(1-42) interfere with gene expression regulated by
activity-dependent signal transduction. Treatment of
cortical neurons with concentrations of A
-(1-42), which did not
affect viability and had no apparent influence on the morphology of the cells, resulted in a marked suppression of
activity-dependent stimulation of CREB and
CREB/CRE-mediated gene transcription, assessed here by evaluating the
expression of one of the CREB target genes, BDNF. Because of the
critical role of CREB and BDNF in synaptic plasticity (13-15, 20),
these observations suggest that low levels of A
-(1-42) can engender
a dysfunctional encoding state in neurons and may initiate early losses
in cognitive function and/or contribute to the propagation of the
cognitive deficit in later stage events of AD as A
continues to
accumulate. In either case, the findings suggest that mechanisms other
than neurodegeneration are contributing to losses in brain function.
Previous studies(e.g. Refs. 1, 8, and 34), including from
our own laboratory, have established that high levels of A
-(1-42)
result in neuronal cell death via apoptosis and secondary necrosis.
Recent studies (9-11) on transgenic mice overexpressing FAD mutant
APPs (1, 4, 5) and observations on AD subjects are consistent with the
view that cognitive decline might occur prior to pronounced
accumulation of A
and before widespread neuronal degeneration takes
place in the brain. Animals overexpressing the FAD mutant APP develop
progressively histopathological abnormalities including typical
neuritic amyloid plaques and dystrophic neurites, but loss of neurons
is not a consistent feature and may relate to the genetic background of
the animals rather than to the phenotype (4, 7, 43). The mice show
functional deficits affecting synaptic plasticity, and the performance
of the aged transgenics in spatial working memory tasks is impaired
(4). In view of our findings, it is particularly important that in
transgenic animals, which were generated in order to increase A
levels in the context of relatively low APP expression (Swedish/Indiana double mutations in APP), deficit in long term potentiation was already
observed at a relatively young age when amyloid plaques are not yet
apparent but an increase in A
expression is detectable (5).
Recent observations have shown that soluble intermediate forms of A
assemblies, such as A
protofilaments (44) and ADDLs (32), can
interfere with neuronal function and survival irrespective of the
formation of mature A
fibrils. Our findings are in agreement with
these results. ADDLs compromised neuron survival, and at sublethal
levels reduced the activity-dependent signaling via CREB at
lower concentrations than fully fibrillar A
, consistent with a role
of the peptide in disrupting neuronal function during the development
of AD.
That low levels of A
-(1-42) may compromise brain functions through
mechanisms unrelated to degenerative changes was supported by our
observation that A
-(25-35) at sublethal concentration did not
interfere with activity-dependent signal transduction and
that caspase inhibitors, which are known to prevent
A
-(1-42)-induced apoptosis, failed to counteract the
A
-(1-42)-evoked suppression of CREB activation.
CREB/CRE is known to play a central and highly conserved role in the
molecular mechanisms underlying synaptic plasticity, including learning
and memory (13-15). As cognitive deficit is central to the
pathophysiology of AD, it is relevant that a reduction of P-CREB levels
has been observed in the postmortem AD brain (45). Our observations
that neuronal activity-induced CREB phosphorylation is suppressed by
sublethal levels of A
-(1-42) are consistent with these findings. It
has been reported, however, that in certain non-neuronal cells A
can
increase CREB phosphorylation. Thus in undifferentiated PC12 cells
A
-(1-40) can lead to CREB activation (46), and in cultures of
primary microglia and THP1 monocytes, relatively high concentration of
fibrillar A
-(25-35) (50 µM) has a similar effect
(47). Responses of these cells to A
are markedly different from
those observed in nerve cells. In contrast to these non-neuronal cells,
our data show that A
-(1-42) had no significant effect on CREB
phosphorylation in resting neurons. Furthermore, in contrast to
A
-(1-42), A
-(25-35) had no significant influence on high
K+-induced activation of CREB. The careful study of
McDonald et al. (47) elucidated that the signal transduction
mechanisms leading to 50 µM A
-(25-35)-induced CREB
phosphorylation in microglia and monocytes involve protein tyrosine
kinase-dependent activation of two parallel pathways,
extracellular signal-regulated kinase and p38 mitogen-activated protein
kinases. On the other hand, in nerve cells the depolarization-induced
phosphorylation of CREB is primarily dependent on Ca2+
influx-mediated activation of
Ca2+-calmodulin-dependent protein kinase IV. This
has been demonstrated previously (17) and confirmed in our studies by
the pronounced inhibition of the high K+-induced CREB
phosphorylation by the Ca2+-calmodulin-dependent protein
kinase inhibitor KN62 (not shown).
The transfection of three familial APP mutations (V642I, V642F, and
V642G) in neurons resulted in suppressed transcriptional activity of
CRE (48). Interference with events downstream of activated CREB was
supported by the finding that sublethal A
-(1-42) suppressed the
neuronal activity-dependent transcription of the exon
III-containing BDNF. BDNF is a complex gene that contains five exons
(42). The coding region is entirely in exon V, whereas each of the
first four exons has a unique promoter, containing different regulatory
elements on the 5'-flanking region. Each of the four 5' exons is,
therefore, differentially regulated and gives rise to four primary BDNF
transcripts. The promoter of exon III contains a CRE and a response
element to a novel Ca2+-regulated factor (17, 18); thus
BDNF is a CREB target gene. The relevance of sublethal
A
-(1-42)-induced suppression of the neuronal
activity-dependent phosphorylation of CREB and the
induction of the CREB target gene BDNF is underlined by the observation that the content of both the BDNF transcript and protein is markedly reduced in brain regions, such as the hippocampus, which are severely affected in AD (23-25). Because of the role of CREB and BDNF in neuronal plasticity, including learning and memory (20, 21), the
reduction may contribute to the cognitive deficit in AD. Although the
BDNF protein is not the exclusive product of the exon III-containing transcript, our finding that sublethal levels of A
-(1-42) result in
the suppression of the activity-dependent phosphorylation
of CREB and the consecutive expression of BDNF indicates that the peptide may interfere with neuronal functions early in the course of
the disease, preceding the stage when massive neuronal degeneration is evident.