From the Department of Neurology, Graduate School of
Medicine, Kyoto University, Kyoto 606-8507, Japan and the
¶ Department of Pharmacology, Graduate School of Pharmaceutical
Sciences, Kyoto University, Kyoto 606-8501, Japan
Received for publication, September 1, 2000, and in revised form, January 12, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Multiple lines of evidence, from molecular
and cellular to epidemiological, have implicated nicotinic transmission
in the pathogenesis of Alzheimer's disease (AD). Here we show the
signal transduction mechanism involved in nicotinic receptor-mediated protection against Alzheimer's disease
(AD)1 is one of the most
common diseases presenting dementia. There are no definitive treatments
or prophylactic agents for this neurodegenerative disease. AD is
characterized by the presence of two types of abnormal deposit, senile
plaques and neurofibrillary tangles, and by extensive neuronal
loss (1). We have found that A In our previous reports, we showed that nicotinic acetylcholine
receptor agonists exert a protective effect against glutamate- and
A In the present study, we showed that, at physiological concentrations,
A Materials--
The sources of drugs and materials used in this
study were as follows: Eagle's minimum essential medium (EMEM) (Nissui
Pharmaceutical Co.); Cell Cultures--
Primary cultures were obtained from the
cerebral cortex of fetal rats (17-19 days gestation) by procedures
described previously (11, 16). Briefly, single cells dissociated from
the cerebral cortex of fetal rats were plated out onto plastic
coverslips placed in Falcon dishes. Cultures were incubated in EMEM
supplemented with 10% fetal calf serum (1 to 5 days after plating out)
or 10% horse serum (6 to 12 days after plating out), glutamine (2 mM), glucose (total 11 mM), NaHCO3
(24 mM), and HEPES (10 mM). Cultures were
maintained at 37 °C in a humidified atmosphere of 5%
CO2. Only mature cultures (10 to 14 days in
vitro) were used for the experiments. The animals were treated in
accordance with the guidelines published in the National Institutes of
Health Guide for the Care and Use of Laboratory Animals.
Treatment of the Cultures--
All experiments were carried out
in EMEM at 37 °C. Cultured neurons were exposed to glutamate for
24 h followed by incubation with EMEM for a further 24 h.
A Assessment of Neurotoxicity--
The number of neurons was
evaluated by immunostaining with anti-MAP2 antibody. Neurotoxicity in
each experiment was defined as a reduction in the survival rate, which
was expressed as percentage survival relative to the survival observed
in control cultures. Immunostaining was performed by the methods
described previously (17), using the primary antibody (anti-MAP2
antibody (diluted 1:500)) for 24 h. At least 200 neurons were
counted in 10 to 20 randomly selected fields at 100 (total
magnification) in control cultures to determine the total number of neurons.
Preparation of Cell Extracts--
Semiconfluent cultures were
exposed to each treatment and incubated at 37 °C for various time
intervals. Subsequently, cells were lysed in a buffer consisting of 20 mM Tris/HCl, pH 7.0, 2 mM EGTA, 25 mM 2-glycerophosphate, 1% Triton X-100, 2 mM
dithiothreitol, 1 mM vanadate, 1 mM
phenylmethylsulfonyl fluoride, and 1% aprotinin and centrifuged at
15,000 × g for 30 min at 4 °C. The supernatants were used as the cell extracts.
Immunoblotting and Immunoprecipitation--
Protein samples in
sodium dodecyl sulfate (SDS) buffer were loaded onto SDS-polyacrylamide
gels. After electrophoresis, proteins were electrotransferred to a
polyvinylidene difluoride membrane (Immobilon, Millipore). Membranes
were incubated with either antibodies in 20 mM Tris/HCl, pH
7.6, 135 mM NaCl, 0.1% Tween 20 containing 5% nonfat dry
milk. Subsequently membranes were incubated with horseradish
peroxidase-conjugated anti-rabbit antibody. Immunoreactive bands were
detected by LumiGLO (New England Biolabs, Inc., Beverly, MA). Protein
samples were immunoprecipitated with antibodies (described above) and
then incubated with protein G-Sepharose as described previously (18).
Samples were then subjected to SDS-polyacrylamide gel electrophoresis
followed by immunoblotting.
Statistics--
Data are expressed as the percentage of
surviving neurons relative to the number of neurons in control culture
(vehicle only) and represent the mean ± S.E. Statistical
significance was determined using one-way analysis of variance followed
by Scheffe's multiple comparisons test.
Pretreatment of Neurons with A
We investigated whether the effect on glutamate cytotoxicity depends
upon the A
In addition, other fragments of A Nicotine Protects Neurons from A
Co-incubating the cultures with nicotine (0.5 µM, 7 days)
and A PI3K Contributes to the Protective Effect of Nicotine against
Glutamate-induced Cytotoxicity--
To investigate the mechanism of
the protective effect of nicotine, we focused on the PI3K cascade
because PI3K has been reported to protect cells from apoptosis through
Akt activation (13). The glutamate toxicity model was adopted because a
low concentration of A
Prolonged exposure to glutamate (50 µM, 24 h)
induced cytotoxicity. Incubating the cultures with nicotine (10 µM) for 24 h prior to glutamate exposure
significantly reduced glutamate cytotoxicity. Simultaneous application
of either LY294002 (10 µM) or wortmannin (100 nM), both PI3K inhibitors, with nicotine reduced the
protective effect of nicotine against glutamate cytotoxicity.
3-(2,4)-Dimethoxybenzylidene anabaseine (DMXB, 10 µM), an
Furthermore, a non-receptor tyrosine kinase inhibitor, PP2 (10 µM), also reduced the protective effect. This implies
that Src is involved in the mechanism of protection. Cycloheximide (1 mg/ml) inhibited the protection, implying that some protein synthesis
is necessary. In contrast, PD98059 (50 µM), a
mitogen-activated protein kinase (MAPK) kinase (MAPKK, also known as
MEK1) inhibitor, did not reduce the protective effect of nicotine
(Figs. 4a and 5).
Nicotine Activates Akt through PI3K Activation and Up-regulates
Bcl-2--
Akt is a serine/threonine protein kinase and a putative
effector of PI3K. When PI3K is activated, it phosphorylates Akt. To investigate the activation of Akt by nicotine through PI3K, we examined
the level of phosphorylated Akt detected by an antibody specific for
phospho-Akt using Western blotting. In preliminary experiments,
phosphorylation of Akt was detected just after the application of
nicotine. The levels of phosphorylated Akt increased and reached a
plateau after around 60-min stimulation and were maintained for 24 h (data not shown). Therefore, 60-min stimulation was adopted for the
following experiments.
Nicotine-induced Akt phosphorylation was blocked by the simultaneous
application of LY294002, showing the involvement of PI3K (Fig.
6a). PD98059 did not alter the
phosphorylating effect of nicotine. Akt phosphorylation was blocked by
Bcl-2 proteins are anti-apoptotic proteins, which can prevent cell
death induced by a variety of toxic insults (22). It has been reported
that Akt activation leads to the overexpression of Bcl-2 (23). Because
nicotine could activate Akt via PI3K, we examined the protein levels of
Bcl-2. Treatment with nicotine for 24 h induced the augmented
level of Bcl-2, and the nicotine-induced up-regulation of Bcl-2 was
reduced by LY294002, indicating the involvement of PI3K signal
transduction (Fig. 6d).
Therefore, we hypothesized that nicotinic receptors may act as
metabotropic receptors, directly transmitting signals to PI3K. In other
words, nicotinic receptors might associate with PI3K. To demonstrate
this, lysates of cortical neurons were immunoprecipitated with a
monoclonal anti- There is still a controversy about the role of A The present data show that only a combination of A We previously showed that nicotinic receptor stimulation protects
neurons from A Our hypothesis for the survival signal transduction is shown in Fig.
8. It is not clear from our experiments
whether other Src family members besides Fyn are associated with -amyloid-enhanced glutamate neurotoxicity. Nicotine-induced protection was suppressed by an
7 nicotinic receptor antagonist (
-bungarotoxin), a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002 and wortmannin), and a Src inhibitor (PP2).
Levels of phosphorylated Akt, an effector of PI3K, and Bcl-2 were
increased by nicotine. The
7 nicotinic receptor was physically
associated with the PI3K p85 subunit and Fyn. These findings indicate
that the
7 nicotinic receptor transduces signals to PI3K in a
cascade, which ultimately contributes to a neuroprotective effect. This
might form the basis of a new treatment for AD.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Amyloid (A
) is a major constituent of senile plaques
and one of the candidates for the cause of the neurodegeneration found in AD, because a negative correlation was found between senile plaques
and neuron density (1). It has been hypothesized that accumulation of
A
precedes other pathological changes and causes neurodegeneration
or neuronal death in vivo (2). Several mutations of the A
precursor protein are found in familial AD, and these mutations are
involved in amyloidogenesis (3). It has also been shown that familial
AD mutations of presenilin 1 enhance the generation of A
1-42 (4).
However, presenilin 1 transgenic mice do not have amyloid plaques in
their brains, possibly because presenilin 1 mutations facilitate
apoptotic neuronal death without plaque formation (5). In addition, it
is controversial whether A
is directly toxic to neurons or not.
25-35 is toxic to neurons and that this
cytotoxicity is inhibited by MK801, an
N-methyl-D-aspartate receptor
antagonist.2 Therefore, we
hypothesized that A
might modulate or enhance glutamate-induced
cytotoxicity. Glutamate, one of the excitotoxic neurotransmitters in
the CNS, can cause intracellular Ca2+ influx, activation of
Ca2+-dependent enzymes such as nitric oxide
(NO) synthase, and production of toxic oxygen radicals leading to cell
death (6). In addition, some reports have shown that A
causes a
reduction in glutamate uptake in cultured astrocytes (7), indicating
that A
-induced cytotoxicity might be mediated via glutamate
cytotoxicity to some extent.
-induced neurotoxicity (8-12). Recently, it has been reported that
activated phosphatidylinositol 3-kinase (PI3K) and Akt kinase promote
neuron survival (13). Anti-apoptotic proteins such as Bcl-2, Bcl-x, and
Bad were thought to be involved in this survival system. Nicotinic
receptors are ionotropic receptors, which allow Ca2+ to
enter cells and function physiologically. It has been shown that the
PI3K cascade is activated by tyrosine kinase or G protein-mediated signals in neuronal cells (14). Conversely, there is no evidence that
nicotinic receptors contain a G protein or tyrosine kinase. However,
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors
are also ionotropic receptors, and it was recently shown that a member
of the Src family, Lyn, is physically associated with AMPA receptors
and mediates signals to PI3K (15). Thus, there is a possibility that
ionotropic receptors such as nicotinic receptors could be associated
with a tyrosine kinase such as Src.
itself is not neurotoxic but enhances the cell death induced by
glutamate. The neuroprotective effect of nicotine was examined,
focusing on the involvement of the PI3K cascade. In addition, we
investigated whether nicotinic receptors function as metabotropic
receptors through any kinase families.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amyloid protein fragments 1-40, 1-42, 40-1,
42-1, 1-16, 12-28 and amyloid P component (27-38) (Bachem);
(
)-nicotine, MK801, and monoclonal anti-
7 nicotinic acetylcholine
antibody (Research Biochemicals International);
-bungarotoxin
(Wako); LY294002 (Biomol Research Laboratories, Inc.); PP2
(Calbiochem); PD98059, anti-phospho- and nonphospho-specific p44/p42
mitogen-activated protein (MAP) kinase and Akt antibodies (New England
Biolabs); anti-microtubule-associated protein 2 (MAP2) antibody
(Sigma); polyclonal anti-Fyn antibody (Upstate Biotechnology Inc.);
polyclonal anti-PI3K p85 subunit antibody and anti-
4 nicotinic
acetylcholine antibody (Santa Cruz); monoclonal anti-PI3K p85 subunit
antibody and anti-Bcl-2 antibody (Transduction Laboratories).
and nicotine were added to the medium before exposure to
glutamate. Antagonists were simultaneously added with nicotine. When
cells were exposed to glutamate, other drugs were removed.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Enhances Glutamate
Cytotoxicity--
Cortical neurons were incubated with A
1-40 (1 nM) or A
1-42 (100 pM) for 7 days, which
did not induce cell death. These are the concentrations of A
in the
cerebrospinal fluid of AD patients (19). Treatment with a low dose of
glutamate (20 µM) alone did not significantly induce cell
death (Fig. 1a). Simultaneous exposure to the same dose of A
1-40 and 1-42 followed by glutamate caused a significant reduction in the number of neuronal cells (Fig.
1a). These findings suggested that A
itself is not
neurotoxic at physiological concentrations but makes neurons vulnerable
to excitatory amino acids.
View larger version (74K):
[in a new window]
Fig. 1.
a, effects of -amyloid
(Beta) on glutamate (Glu) neurotoxicity. Cultures
were exposed to A
1-40 (1 nM) and/or A
1-42 (100 pM) for 7 days followed by a 24-h incubation with 20 µM glutamate. Neither peptide alone caused neuronal
death. Simultaneous application of A
1-40 and A
1-42
(Beta 1-40/1-42) did not cause neuronal death, but
enhanced glutamate cytotoxicity. n = 4; *,
p < 0.01 compared with glutamate alone. b,
immunostained images showing A
-enhanced glutamate neurotoxicity.
Panel A, control; panel B, 20 µM
glutamate alone; panel C, A
1-40 (1 nM) + A
1-42 (100 pM); panel D, A
1-40 (1 nM) + A
1-42 (100 pM) + glutamate (20 µM). Bar = 1 × 10
4
m
structure. Scrambled A
, A
40-1, or 42-1 did not
enhance glutamate cytotoxicity, even when incubated simultaneously with
other peptides including 1-40 or 1-42 (Fig.
2a). None of the peptides
caused neuronal death when administered alone.
View larger version (25K):
[in a new window]
Fig. 2.
a, effects of scrambled beta amyloid
(Beta) on glutamate (Glu) neurotoxicity. Cultures
were exposed to A 1-40 (1 nM), A
40-1 (1 nM), A
1-42 (100 pM), or A
42-1 (100 pM) for 7 days followed by a 24-h incubation with 20 µM glutamate. None of the peptides alone caused neuronal
death. n = 4; *, p < 0.01 compared
with glutamate alone. b, effects of other fragments of
-amyloid (Beta) on glutamate (Glu)
neurotoxicity. Cultures were exposed to A
1-40 (1 nM),
A
1-42 (100 pM), A
1-16 (10 nM), A
12-28 (10 nM), or A
27-38 (10 nM) for 7 days followed by a 24-h incubation with 20 µM glutamate.
Combined administration of fragments was performed as shown in the
figure. None of the peptides alone caused neuronal death.
n = 4; *, p < 0.01 compared with
glutamate alone.
, such as A
1-16 (10 nM), 12-28 (10 nM), and the amyloid P
component (27-38) (10 nM), did not significantly reduce
the number of viable neurons after exposure to glutamate (Fig.
2b). None of the peptides caused neuronal death when
administered alone (data not shown). Although A
1-16 enhanced glutamate cytotoxicity, this was not statistically significant.
-enhanced Glutamate
Cytotoxicity--
We used the 1-40 and 1-42 fragments of A
,
because these fragments are found in the brains of AD patients and are
the most potent combination for the enhancement of glutamate
cytotoxicity. Cortical neurons were incubated with both A
1-40 (1 nM) and A
1-42 (100 pM) for 7 days, which
enhanced glutamate cytotoxicity. This effect was inhibited by MK801
when incubated with glutamate, indicating that the toxicity was
mediated via N-methyl-D-aspartate receptors
(Fig. 3).
View larger version (12K):
[in a new window]
Fig. 3.
Effects of nicotine (Nic)
on -amyloid (Beta)-enhanced
glutamate (Glu) neurotoxicity. Cultures were
exposed to A
1-40 (1 nM) and A
1-42 (100 pM) for 7 days followed by a 24-h incubation with 20 µM glutamate. Toxicity caused by this treatment was
inhibited by 1 µM MK801 (MK). Nicotine
protected cells from A
-enhanced glutamate neurotoxicity.
n = 4; *, p < 0.01 compared with
glutamate alone; **, p < 0.01 compared with glutamate + A
.
significantly reduced A
-enhanced glutamate cytotoxicity (Fig. 3). Previously, we reported that nicotine protects neurons from
glutamate-induced cytotoxicity (8-10). Therefore, we hypothesized that
the protective effect of nicotine against A
-enhanced glutamate cytotoxicity depends upon its effect on glutamate toxicity.
alone was not toxic or only enhanced
glutamate toxicity. Furthermore, we showed that nicotine did not
directly affect the A
conformation as described previously (20).
-Bungarotoxin (
BTX, 1 nM), an
7-selective
nicotinic receptor antagonist, also blocked the protection, indicating
that the
7 subtype of nicotinic receptors is involved in this effect
(Figs. 4a and 5).
View larger version (16K):
[in a new window]
Fig. 4.
a, effect of nicotine on
glutamate-induced cytotoxicity. Cultures were exposed to glutamate (50 µM) for 24 h followed by incubation in EMEM for
24 h, which induced cell death. Nicotine (1 µM) was
preincubated with the cultures for 24 h. If used, LY294002
(LY), wortmannin (Wort), -bungarotoxin
(BTX), PP2, cycloheximide (Cyclo), and PD98059
(PD) were added to the medium containing nicotine. After the
preincubation, medium was replaced by glutamate-containing
medium for 24 h and finally replaced with EMEM, as
described above. LY294002, wortmannin, and PI3K inhibitors all
significantly reduced the protective effect of nicotine. Furthermore,
PP2, a Src family tyrosine kinase inhibitor, also reduced the
protective effect of nicotine. n = 4; *,
p < 0.01 compared with glutamate alone; **,
p < 0.01 compared with glutamate + nicotine.
NS, not significant. b, effect of DMXB on
glutamate-induced cytotoxicity. DMXB (10 µM) was added to
the medium and incubated for 24 h. LY294002 significantly reduced
the protective effect. n = 4; *, p < 0.05 compared with glutamate alone; **, p < 0.05 compared with glutamate + nicotine.
7-selective nicotinic receptor agonist
(21), also exerted a protective effect against glutamate-induced
cytotoxicity. This effect was inhibited by 1 nM
BTX,
indicating that the effect of DMXB is mediated via
7 receptors.
Furthermore, the protection was also reduced by 10 µM
LY294002 (Fig. 4b). From these findings, we concluded that
7 nicotinic receptor stimulation exerts a neuroprotective effect
against glutamate cytotoxicity and that the PI3K cascade is involved in
this effect. We previously showed that
4
2 subunit nicotinic
receptor stimulation also exerted a protective effect against A
- and
glutamate-induced cytotoxicity (10, 12). This effect, however, was not
inhibited by LY294002 (data not shown).
View larger version (118K):
[in a new window]
Fig. 5.
Immunostained images showing the protective
effect of nicotine against glutamate neurotoxicity. A,
control; B, 50 µM glutamate alone;
C, nicotine (10 µM) + glutamate; D,
nicotine + LY294002 (10 µM) + glutamate; E,
nicotine + BTX (1 nM) + glutamate; F,
nicotine + PD98059 (50 µM) + glutamate.
Bar = 1 × 10
4 m
BTX, indicating that the phosphorylating effect of nicotine is
mediated by
7 nicotinic receptors (Fig. 6, b and
c). Conversely, dihydro-
-erythroidine (100 nM), a selective
4
2 nicotinic receptor antagonist,
did not block nicotine-induced Akt phosphorylation (Fig.
6c). PP2 blocked Akt phosphorylation, indicating the
involvement of tyrosine kinase. 10 µM MK801 did not block
Akt phosphorylation, showing that the secondary release of glutamate
has no effect (Fig. 6b).
View larger version (33K):
[in a new window]
Fig. 6.
Representative data of phosphorylation of
Akt/PKB by nicotine detected by Western blot using antibody specific
for phosphorylated Akt. a, nicotine (Nic, 10 µM) increased the levels of the phosphorylated Akt
compared with the total Akt levels. This phosphorylation was
significantly inhibited by LY294002 (10 µM). PD98059 (50 µM), a MEK1 inhibitor, did not interfere with Akt
phosphorylation. Each inhibitor was added simultaneously with
glutamate. PD, PD98059; LY, LY294002.
n = 6. b, Akt phosphorylation was inhibited
by BTX (1 nM) and PP2 (10 µM), a Src
inhibitor. MK801 (10 µM) did not reduce the
phosphorylation of Akt induced by nicotine. BTX,
BTX;
MK, MK801. n = 6. c, Akt
phosphorylation was not inhibited by dihydro-
-erythroidine (100 nM) in contrast to
BTX. BTX,
BTX.
n = 6. d, Bcl-2 was up-regulated after
nicotine treatment. LY294002 (10 µM) inhibited the
up-regulation of Bcl-2, indicating that nicotine-induced up-regulation
of Bcl-2 is mediated via the PI3K cascade. n = 6.
7 Nicotinic Acetylcholine Receptors Physically Associate with
PI3K and Fyn--
The present results show that nicotine protects
neurons from glutamate cytotoxicity by activating PI3K, which activates
Akt and up-regulates Bcl-2. Nicotinic receptors are ionotropic, and there have been no reports of ionotropic receptors activating PI3K directly.
7 nicotinic receptor antibody. Immunoprecipitated (IP) samples were then separated by SDS-polyacrylamide gel
electrophoresis and stained with a polyclonal anti-PI3K p85 subunit
antibody using the Western blot technique. The PI3K p85 subunit was
detected in this IP sample, indicating that
7 nicotinic receptors
bind to the PI3K p85 subunit (Fig.
7a). Conversely, lysate
immunoprecipitated with the polyclonal anti PI3K p85 subunit antibody
contained protein detected by the monoclonal anti-
7 nicotinic
receptor antibody. Non-receptor type tyrosine kinase, Fyn, was also
co-immunoprecipitated with the
7 nicotinic receptors (Fig.
7b). The
4 subunit of the nicotinic receptor was also
investigated and was not detected in lysates immunoprecipitated with
the PI3K p85 subunit, Fyn, or the
7 nicotinic receptor antibody
(Fig. 7c).
View larger version (27K):
[in a new window]
Fig. 7.
Association of the 7
nicotinic receptor with PI3K and Fyn. a, the PI3K was
detected in IP produced using the anti-
7 nicotinic receptor antibody
(left). Conversely, the
7 nicotinic receptor was detected
in IP produced using the anti-p85 subunit PI3K antibody
(right). n = 6. nAChR, nicotinic
acetylcholine receptor. b, Fyn was detected in IP produced
using the anti-
7 nicotinic receptor antibody. Conversely,
7
nicotinic receptors were detected in IP produced using the anti-Fyn
antibody (right). n = 6. c, the
4 subunit of the nicotinic receptor was not detected in any of the
IP samples produced using the anti-PI3K p85 subunit, Fyn, or the
7
nicotinic receptor antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and glutamate
in the pathogenesis of AD. However, amyloid accumulation is one of the
earliest changes in AD pathology, and this peptide may cause neuronal
death in the central nervous system (2). The precise mechanism of
A
-induced cytotoxicity remains unknown, although various hypotheses
have been suggested. Oxidative stress, or free radical generation, is
one of the candidates for the cause of A
-induced cytotoxicity.
Previous reports have shown that A
stimulates NO production through
Ca2+ entry triggered by activated
N-methyl-D-aspartate-gated channels (24). Other
reports have suggested that A
inhibits glutamate uptake and causes
extracellular glutamate increase (7). There are also some reports that
have proposed that A
enhances the toxicity induced by excitotoxins
(25, 26). These reports implied that A
-induced cytotoxicity might
be, at least in part, mediated via glutamate toxicity. The present
study also indicated that A
enhances glutamate neurotoxicity.
1-40 and A
1-42 enhanced glutamate cytotoxicity. The concentration of these
peptides used in this study were almost the same level as that detected
in the cerebrospinal fluid of AD patients (19). Other fragments did not
enhance the toxicity, even when administered simultaneously with A
1-40 or A
1-42. Therefore, the enhancing effect appears to be
related to the structure of the peptides so that the combination of
A
1-40 and A
1-42 might play a specific role in making neurons
vulnerable to glutamate. The full length of both peptides appears to be necessary.
- and glutamate-induced cell death (8-12). In the
present study, we showed that nicotinic receptor stimulation, especially
7 receptor stimulation, inhibits glutamate toxicity and
that PI3K-Akt signal transduction contributes to this effect. In
addition, the Bcl-2 family is stimulated downstream of the PI3K-Akt
cascade and works as an anti-neuronal death factor. It is proposed that
PI3K-Akt activation promotes cell survival, and up-regulation of Bcl-2
is one of the major reasons for cell survival (23, 27). Nicotinic
receptor stimulation transduces these survival signals besides its role
as a transmitter. The
sheet conformation of A
might influence
its function, such as toxicity or modulation of survival signals.
However, in our experiments, nicotine and nicotinic agonists did not
influence the
sheet conformation (20). Instead, signal transduction
was shown to be important for the protective effect of nicotine.
7
receptors. However, a relationship between nicotinic receptors and Fyn
was implicated because catecholamine release induced by nicotine is dependent upon the presence of Fyn and extracellular Ca2+,
and no other Src member was detected (28). In our preliminary data,
removal of extracellular Ca2+ suppressed Akt
phosphorylation induced by nicotine (data not shown). We showed that an
inhibitor of Src tyrosine kinase reduced Akt phosphorylation. In
addition, PI3K and Fyn are physically associated with
7 nicotinic
receptors. Therefore, nicotinic receptor stimulation might
phosphorylate Akt by signal transduction through Fyn to PI3K, and
extracellular Ca2+ might contribute to this process.
View larger version (22K):
[in a new window]
Fig. 8.
A schematic model of the neuroprotection
induced by 7 nicotinic receptor
stimulation.
In the brain, nicotinic receptors include several subtypes with
differing properties and functions. The abundant presence of 7
receptors in the hippocampus, neocortex, and basal ganglia (29), in
conjunction with the memory-enhancing activity of selective
7
nicotinic agonists such as DMXB (30), suggests a significant role for
7 receptors in learning and memory. In addition, the protective
action of nicotine is mediated, at least partially, through
7
receptors. Recently it was reported that A
1-42 binds to
7
receptors (31), and this might inhibit
7 nicotinic
receptor-dependent learning and memory. The reduction of
7 receptor activation might cause neurons vulnerable to various
toxic insults such as glutamate. In our study, however, the lysate
immunoprecipitated with anti-
7 antibody did not contain A
1-42
(data not shown). This might be because the antibody we used was
different from that used in the report (31), but we could not prove
that enhancement of glutamate toxicity depends upon the reduction of
7 nicotinic receptors. In addition, A
12-28, which suppresses
the formation of the
7-A
complex (31), did not inhibit the
enhanced glutamate toxicity induced by the combined exposure to of A
1-40 and A
1-42 (Fig. 2b). Therefore, it is unlikely
that the protective effect of nicotine depends upon the displacement of
7-A
binding.
Recently, it was shown that ionotropic receptors have properties similar to metabotropic receptors. AMPA receptors are physically associated with a member of the Src family, Lyn (15). The AMPA receptor activates Lyn, which then activates MAPK. Through the Lyn-MAPK pathway, AMPA receptors generate intracellular signals and transmit them from the cell surface to the nucleus. Nicotinic receptors are known to be ionotropic receptors. The present study indicated that nicotinic receptors also have metabotropic properties, which contribute to neuronal survival. It is likely, however, that many unrecognized receptor functions still remain.
The cholinergic system is affected in dementia-causing diseases, AD
among others, and a reduction in the number of nicotinic receptors in
these diseases has been reported (32, 33). It is of interest that
down-regulation of nicotinic receptors can result in neuronal cell
death or neurodegeneration (34). Nicotine might function not only as a
cholinergic agonist but also as a neuroprotective agent. Our present
study suggests that nicotinic receptor stimulation could protect
neurons from A-enhanced glutamate toxicity. Thus, by an early
diagnosis of AD and protective therapy with nicotinic receptor
stimulation, we could delay the progress of AD.
![]() |
ACKNOWLEDGEMENT |
---|
We thank the Taiho Pharmaceutical Co., for providing us with DMXB.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture and grants from the Ministry of Welfare of Japan, the Smoking Research Foundation, and the Inamori Foundation.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.
§ To whom correspondence should be addressed: Dept. of Neurology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan. Tel.: 81-75-751-3767; Fax: 81-75-751-9541; E-mail: i53367@sakura.kudpc.kyoto-u.ac.jp.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M008035200
2 T. Kihara, S. Shimohama, H. Sawada, K. Honda, T. Nakamizo, H. Shibasaki, T. Kume, and A. Akaike, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
AD, Alzheimer's
disease;
A,
-amyloid;
NO, nitric oxide;
PI3K, phosphatidylinositol 3-kinase;
AMPA,
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid;
EMEM, Eagle's
minimum essential medium;
MAP2, microtubule-associated protein 2;
BTX,
-bungarotoxin;
DMXB, 3-(2,4)-dimethoxybenzylidene
anabaseine;
MAPK, mitogen-activated protein kinase;
IP, immunoprecipitate(s).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Giannakopoulos, P., Hof, P. R., Kovari, E., Vallet, P. G., Herrmann, F. R., and Bouras, C. (1996) J. Neuropathol. Exp. Neurol. 55, 1210-1220[Medline] [Order article via Infotrieve] |
2. | Yankner, B. A., Duffy, L. K., and Kirschner, D. A. (1990) Science 250, 279-282[Medline] [Order article via Infotrieve] |
3. | Citron, M., Oltersdorf, T., Haass, C., McConlogue, L., Hung, A. Y., Seubert, P., Vigo, P. C., Lieberburg, I., and Selkoe, D. J. (1992) Nature 360, 672-674[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Tomita, T.,
Maruyama, K.,
Saido, T. C.,
Kume, H.,
Shinozaki, K.,
Tokuhiro, S.,
Capell, A.,
Walter, J.,
Grunberg, J.,
Haass, C.,
Iwatsubo, T.,
and Obata, K.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2025-2030 |
5. | Guo, Q., Fu, W., Sopher, B. L., Miller, M. W., Ware, C. B., Martin, G. M., and Mattson, M. P. (1999) Nat. Med. 5, 101-106[CrossRef][Medline] [Order article via Infotrieve] |
6. | Tamura, Y., Sato, Y., Akaike, A., and Shiomi, H. (1992) Brain Res. 592, 317-325[Medline] [Order article via Infotrieve] |
7. | Harris, M. E., Wang, Y., Pedigo, N. W. J., Hensley, K., Butterfield, D. A., and Carney, J. M. (1996) J. Neurochem. 67, 277-286[Medline] [Order article via Infotrieve] |
8. | Akaike, A., Tamura, Y., Yokota, T., Shimohama, S., and Kimura, J. (1994) Brain Res. 644, 181-187[CrossRef][Medline] [Order article via Infotrieve] |
9. | Shimohama, S., Akaike, A., and Kimura, J. (1996) Ann. N. Y. Acad. Sci. 777, 356-361[Abstract] |
10. | Kaneko, S., Maeda, T., Kume, T., Kochiyama, H., Akaike, A., Shimohama, S., and Kimura, J. (1997) Brain Res. 765, 135-140[CrossRef][Medline] [Order article via Infotrieve] |
11. | Kihara, T., Shimohama, S., Sawada, H., Kimura, J., Kume, T., Kochiyama, H., Maeda, T., and Akaike, A. (1997) Ann. Neurol. 42, 159-163[Medline] [Order article via Infotrieve] |
12. | Kihara, T., Shimohama, S., Urushitani, M., Sawada, H., Kimura, J., Kume, T., Maeda, T., and Akaike, A. (1998) Brain Res. 792, 331-334[CrossRef][Medline] [Order article via Infotrieve] |
13. |
del Peso, L.,
Gonzalez-Garcia, M.,
Page, C.,
Herrera, R.,
and Nunez, G.
(1997)
Science
278,
687-689 |
14. |
Perkinton, M. S.,
Sihra, T. S.,
and Williams, R. J.
(1999)
J. Neurosci.
19,
5861-5874 |
15. | Hayashi, T., Umemori, H., Mishina, M., and Yamamoto, T. (1999) Nature 397, 72-76[CrossRef][Medline] [Order article via Infotrieve] |
16. | Shimohama, S., Ogawa, N., Tamura, Y., Akaike, A., Tsukahara, T., Iwata, H., and Kimura, J. (1993) Brain Res. 632, 296-302[Medline] [Order article via Infotrieve] |
17. | Sawada, H., Ibi, M., Kihara, T., Urushitani, M., Akaike, A., Kimura, J., and Shimohama, S. (1998) Ann. Neurol. 44, 110-119[Medline] [Order article via Infotrieve] |
18. | Kume, T., Kochiyama, H., Kaneko, S., Maeda, T., Kaneko, S., Akaike, A., Shimohama, S., Kihara, T., Kimura, J., Wada, K., and Koizumi, S. (1997) Brain Res. 756, 200-204[CrossRef][Medline] [Order article via Infotrieve] |
19. | Jensen, M., Schroder, J., Blomberg, M., Engvall, B., Pantel, J., Ida, N., Basun, H., Wahlund, L. O., Werle, E., Jauss, M., Beyreuther, K., Lannfelt, L., and Hartmann, T. (1999) Ann. Neurol. 45, 504-511[CrossRef][Medline] [Order article via Infotrieve] |
20. | Kihara, T., Shimohama, S., and Akaike, A. (1999) Jpn. J. Pharmacol. 79, 393-396[CrossRef][Medline] [Order article via Infotrieve] |
21. | Hunter, B. E., de Fiebre, C. M., Papke, R. L., Kem, W. R., and Meyer, E. M. (1994) Neurosci. Lett. 168, 130-134[CrossRef][Medline] [Order article via Infotrieve] |
22. | Zhong, L. T., Kane, D. J., and Bredesen, D. E. (1993) Mol. Brain Res. 19, 353-355[Medline] [Order article via Infotrieve] |
23. | Matsuzaki, H., Tamatani, M., Mitsuda, N., Namikawa, K., Kiyama, H., Miyake, S., and Tohyama, M. (1999) J. Neurochem. 73, 2037-2046[Medline] [Order article via Infotrieve] |
24. | O'Mahony, S., Harkany, T., Rensink, A. A., Abraham, I., De Jong, G. I., Varga, J. L., Zarandi, M., Penke, B., Nyakas, C., Luiten, P. G., and Leonard, B. E. (1998) Brain Res. Bull. 45, 405-411[CrossRef][Medline] [Order article via Infotrieve] |
25. | Dornan, W. A., Kang, D. E., McCampbell, A., and Kang, E. E. (1993) Neuroreport 5, 165-168[Medline] [Order article via Infotrieve] |
26. | Morimoto, K., Yoshimi, K., Tonohiro, T., Yamada, N., Oda, T., and Kaneko, I. (1998) Neuroscience 84, 479-487[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Eves, E. M.,
Xiong, W.,
Bellacosa, A.,
Kennedy, S. G.,
Tsichlis, P. N.,
Rosner, M. R.,
and Hay, N.
(1998)
Mol. Cell. Biol.
18,
2143-2152 |
28. | Allen, C. M., Ely, C. M., Juaneza, M. A., and Parsons, S. J. (1996) J. Neurosci. Res. 44, 421-429[CrossRef][Medline] [Order article via Infotrieve] |
29. | Clarke, P. B., Schwartz, R. D., Paul, S. M., Pert, C. B., and Pert, A. (1985) J. Neurosci. 5, 1307-1315[Abstract] |
30. | Meyer, E. M., Tay, E. T., Papke, R. L., Meyers, C., Huang, G. L., and de Fiebre, C. M. (1997) Brain Res. 768, 49-56[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Wang, H. Y.,
Lee, D. H.,
D'Andrea, M. R.,
Peterson, P. A.,
Shank, R. P.,
and Reitz, A. B.
(2000)
J. Biol. Chem.
275,
5626-5632 |
32. | Shimohama, S., Taniguchi, T., Fujiwara, M., and Kameyama, M. (1986) J. Neurochem. 46, 288-293[Medline] [Order article via Infotrieve] |
33. | Whitehouse, P. J., and Kalaria, R. N. (1995) Alzheimer Dis. Assoc. Disord. 9, 3-5[Medline] [Order article via Infotrieve] |
34. |
Zoli, M.,
Picciotto, M. R.,
Ferrari, R.,
Cocchi, D.,
and Changeux, J. P.
(1999)
EMBO J.
18,
1235-1244 |