Activation of Muscarinic Receptors Inhibits
-Amyloid
Peptide-induced Signaling in Cortical Slices*
Zhenglin
Gu,
Ping
Zhong, and
Zhen
Yan
From the Department of Physiology and Biophysics, State University
of New York at Buffalo, School of Medicine and Biomedical Sciences,
Buffalo, New York 14214
Received for publication, September 26, 2002, and in revised form, January 31, 2003
 |
ABSTRACT |
Deposition of fibrillar aggregates of the
-amyloid peptide (A
) is a key pathologic feature during the early
stage of Alzheimer's disease. The initial neuronal responses to
A
in cortical circuits and the regulation of A
-induced signaling
remain unclear. In this study, we found that exposure of cortical
slices to A
1-42 or A
25-35 induced
a marked increase in the activation of protein kinase C (PKC) and
Ca2+/calmodulin-dependent kinase II (CaMKII),
two enzymes critically involved in a variety of cellular functions.
Activation of M1 muscarinic receptors, but not nicotinic receptors,
significantly inhibited the A
activation of PKC and CaMKII.
Increasing inhibitory transmission mimicked the M1 effect on A
,
whereas blocking GABAA receptors eliminated the M1 action.
Moreover, electrophysiological evidence shows that application of A
to cortical slices induced action potential firing and enhanced
excitatory postsynaptic currents, whereas muscarinic agonists potently
increased inhibitory postsynaptic currents. These results suggest that
A
activates PKC and CaMKII through enhancing excitatory activity in
glutamatergic synaptic networks. Activation of M1 receptors inhibits
A
signaling by enhancing the counteracting
GABAergic inhibitory transmission. Thus the
muscarinic reversal of the A
-induced biochemical and physiological
changes provides a potential mechanism for the treatment of
Alzheimer's disease with cholinergic enhancers.
 |
INTRODUCTION |
The 40-42-amino acid
-amyloid peptide
(A
)1 is a major
constituent of senile plaques (1), extracellular protein aggregates that are used as a histopathological hallmark for the diagnosis of
Alzheimer's disease (AD). Emerging evidence has suggested that A
makes a direct contribution to the pathogenesis of AD (2, 3). A
peptides are produced by the cleavage of
-amyloid precursor protein
(APP) (4). Mutations in the APP gene increases the rate of cleavage,
thereby leading to the overproduction of A
(5, 6). Transgenic mice
overexpressing mutant APP genes exhibit AD-like A
deposits and
cognition impairments (7-9). In vitro studies in cell lines
and cultured neurons show that fibrillar A
is neurotoxic at high
concentrations (10, 11), and most strikingly, A
exposure renders
neurons more vulnerable to excitatoxicity (12, 13). Although intensive
efforts have been concentrated on factors affecting A
production,
aggregation, and metabolism (14, 15), little is known about the
earliest biochemical and physiological changes in neurons in response
to the subtoxic concentrations of A
, which may be critical for
subsequent neurodegenerative changes and the factors that can regulate
the A
-initiated signaling.
In addition to A
deposits, a prominent feature of AD is the
degeneration of basal forebrain cholinergic neurons and ensuing deficient cholinergic functions in their target areas including cortex
and hippocampus (16-18). Despite an improved understanding of the
critical role of the cholinergic system in normal cognition and
dementia (19, 20), it has been largely unclear how cholinergic deficits
and A
accumulation might be related to each other. Previous evidence
suggests that A
can induce cholinergic hypofunction independently of
apparent neurotoxicity (21), whereas activation of muscarinic receptors
can inhibit the generation of amyloidogenic A
(22, 23), indicative
of a reciprocal negative regulation between them.
Here we report that treatment of cortical slices with A
led to a
rapid increase in the activation of protein kinase C (PKC) and
Ca2+/calmodulin-dependent kinase II (CaMKII),
and this effect was dependent on A
-induced excitation of
glutamatergic synapses and ensuing Ca2+ influx.
Furthermore, the A
signaling was selectively down-regulated by the
activation of muscarinic receptors through a mechanism involving
enhanced GABAergic inhibition. Given the key roles of PKC and CaMKII in
regulating a wide range of neuronal functions from synaptic plasticity
to cell survival (24, 25), their strong activation by A
could
interfere with these critical processes and thereby might contribute to
cognitive deficits in AD. The ability of muscarinic receptors to block
the A
signaling provides one potential mechanism supporting the
notion that enhancing cholinergic transmission could be an effective
therapeutic strategy in AD treatment (26, 27).
 |
MATERIALS AND METHODS |
Western Blot Analysis--
Young adult (3-5 weeks postnatal)
rat slices containing frontal cortex were prepared as previously
described (28, 29). All of the experiments were carried out according
to the Yroval of State University of New York at Buffalo Animal Care
Committee. In brief, the rats were anesthetized by inhaling
2-bromo-2-chloro-1,1,1-trifluoroethane (1 ml/100 g; Sigma) and
decapitated; the brains were quickly removed, iced, and then blocked
for slicing. The blocked tissue was cut in 300-400-µm slices with a
Vibrotome while being bathed in a low Ca2+ (100 µM), HEPES-buffered salt solution (140 mM
sodium isethionate, 2 mM KCl, 4 mM
MgCl2, 0.1 mM CaCl2, 23 mM glucose, 15 mM HEPES, 1 mM
kynurenic acid, pH 7.4, osmolarity = 300-305). The slices were
then incubated for 1 h at room temperature (20-22 °C) in a
NaHCO3-buffered saline bubbled with 95% O2,
5% CO2 (126 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2,
26 mM NaHCO3, 1.25 mM
NaH2PO4, 10 mM glucose, 1 mM pyruvic acid, 0.05 mM glutathione, 0.1 mM NG-nitro-L-arginine,
1 mM kynurenic acid, pH 7.4; osmolarity = 300-305). All of
the reagents were obtained from Sigma.
For detecting activated PKC, a phospho-PKC (pan) antibody that
recognizes PKC
,
I,
II,
,
, and
isoforms only when phosphorylated at a carboxyl-terminal residue
homologous to Ser660 of PKC
II was used in
the Western blot analysis. Activated CaMKII was detected with an
antibody recognizing the Thr286-phosphorylated CaMKII.
After incubation, the slices were transferred to boiling 1% SDS and
homogenized immediately. Insoluble material was removed by
centrifugation (13,000 × g for 10 min), and the protein concentration for each sample was measured. To minimize slice
variations, pairs of coronal sections were compared. Equal amounts of
protein from slice homogenates were separated on 7.5% acrylamide gels
and transferred to nitrocellulose membranes. The blots were blocked
with 5% nonfat dry milk for 1 h at room temperature. Then the
blots were incubated with the phospho-PKC (pan) antibody (Cell
Signaling, 1:2000) or the anti-Thr286-phosphorylated CaMKII
antibody (Promega, 1:2,000) for 1 h at room temperature. After
being rinsed, the blots were incubated with horseradish
peroxidase-conjugated anti-rabbit antibodies (Amersham Biosciences;
1:2000) for 1 h at room temperature. Following three washes, the
blots were exposed to the enhanced chemiluminescence substrate. Then
the blots were stripped for 1 h at 50 °C followed by saturation
in 5% nonfat dry milk and incubated with a PKC antibody (Santa Cruz,
1:2000) recognizing the
,
, and
isoforms or an anti-CaMKII
antibody (Upstate Biotechnology Inc.; 1:2000). Quantitation was
obtained from densitometric measurements of immunoreactive bands on
autoradiograms. The data correspond to the means ± S.D. of 5-10
samples/condition and were analyzed by ANOVA tests.
Cholinergic receptor ligands oxotremorine methiodide (oxo-M),
pirenzepine, methoctramine, nicotine, as well as channel blockers or
activators (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]
cyclohepten-5,10-imine maleate (MK-801),
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), D(
)-2-amino-5-phosphonopetanoic acid (D-APV), nifedipine,
diazepam (Sigma) were made up as concentrated stocks in water and
stored at
20 °C. The selective ligand for M1 receptors, muscarinic
toxin 7 (MT-7), was obtained from Peptides International (Louisville, KY). The stocks were thawed and diluted immediately prior to use. The
-amyloid peptide A
25-35, the control peptide
containing the reverse sequence A
35-25, the full-length
peptide A
1-42, A
1-40, and the control
peptide containing the reverse sequence A
40-1 were
obtained from Sigma. A
25-35 and A
35-25 were resuspended in sterile distilled water at a concentration of 2 mM and incubated at 37 °C for 1 h to allow fibril
formation (30). A
1-42, A
1-40, and
A
40-1 were resuspended in 50% phosphate-buffered
saline at a concentration of 1.5 mg/ml and incubated at 37 °C for
4-7 days to allow fibril formation (31).
Protein Kinase Assay--
After incubation, the brain slices
were lysed in cold lysis buffer (1% Triton X-100, 5 mM EDTA, 10 mM Tris, 50 mM NaCl, 30 mM
Na4P2O7·10H2O, 50 mM NaF, 0.1 mM Na3VO4,
1 mM phenylmethylsulfonyl fluoride, Complete protease
inhibitors from Roche Applied Science) on ice for 30 min. The brain
lysates were centrifuged and ultracentrifuged, and PKC was
immunoprecipitated with mouse monoclonal anti-PKC

(Santa Cruz
Biotechnology, Santa Cruz, CA) for 1 h, followed by the addition
of 50 µl of protein A-Sepharose beads and incubation for 1 h at
4 °C. The beads were pelleted by centrifugation and washed three
times with lysis buffer and three times with kinase buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2)
and then resuspended in 30 µl of kinase buffer. In vitro
kinase activity was measured in the PKC immunoprecipitates using myelin
basic protein as substrate. The assay was initiated by the
addition of 1 µl of [
-32P]ATP (10 mCi/ml), 4 µl of
ATP (100 µM), and 1 µl of myelin basic protein (5 mg/ml), continued for 20 min at room temperature, and stopped by
boiling samples in SDS/PAGE sample buffer. The samples were loaded onto
a 20% polyacrylamide gel and subjected to electrophoresis. The gels
were vacuum-dried and exposed to Biomax film. The kinase activity was
quantified by PhosphorImager.
Electrophysiological Recordings in Slices--
To evaluate the
regulation of neuronal excitability by A
25-35 in
frontal cortical slices, the whole cell current clamp recordings were
used with patch electrodes (5-9 M
) filled with the following
internal solution 60 mM
K2SO4, 60 mM
N-methyl-D-glucamine, 40 mM HEPES, 4 mM MgCl2, 5 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), 12 mM phosphocreatine, 2 mM Na2ATP, 0.2 mM
Na3GTP, 0.1 mM leupeptin, pH 7.2-7.3
(osmolarity = 265-270). For the measurement of spontaneous excitatory
postsynaptic currents (sEPSCs) in slices, the whole cell voltage clamp
recordings were used with the following internal solution: 130 mM cesium methanesulfonate, 10 mM CsCl, 4 mM NaCl, 10 mM HEPES, 1 mM
MgCl2, 5 mM EGTA, 12 mM
phosphocreatine, 5 mM MgATP, 0.2 mM
Na3GTP, 0.1 mM leupeptin, pH 7.2-7.3
(osmolarity = 265-270). The GABAA receptor antagonist
bicuculline (10 µM) was added to the recording solution
to block the inhibitory transmission. For the measurement of
spontaneous inhibitory postsynaptic currents (sIPSCs), the whole cell
voltage clamp internal solution contained 100 mM CsCl, 30 mM N-methyl-D-glucamine, 10 mM HEPES, 1 mM MgCl2, 4 mM NaCl, 5 mM EGTA, 12 mM
phosphocreatine, 2 mM MgATP, 0.2 mM Na3GTP, 0.1 mM leupeptin, pH 7.2-7.3 (265-270
mosmol/liter). The AMPA/KA receptor antagonist CNQX (10 µM) and NMDA receptor antagonist D(
)-2-amino-5-phosphonopetanoic acid (25 µM) were added to the recording solution to block
glutamate transmission. The slice (300 µm) was placed in a perfusion
chamber attached to the fixed stage of an upright microscope (Olympus)
and submerged in continuously flowing oxygenated artificial
cerebrospinal fluid. The cells were visualized with a 40× water
immersion lens and illuminated with near infrared light, and the image
was detected with an infrared-sensitive CCD camera. A Multiclamp 700A
amplifier (Axon Instruments, Union City, CA) was used for these
recordings. Tight seals (2-10 G
) from visualized pyramidal neurons
were obtained by applying negative pressure. The membrane was disrupted
with additional suction, and the whole cell configuration was obtained.
The access resistances ranged from 13 to 18 M
and were compensated
50-70%. The cells were held at
70 mV for the recording of
spontaneous EPSCs or IPSCs. The Mini Analysis Program (Synaptosoft,
Leonia, NJ) was used to analyze synaptic activity. Synaptic currents of
1 min (200-1000 events) under each different treatment were used for analysis. Statistical comparisons of the synaptic currents were made
using the Kolmogorov-Smirnov (K-S) test.
 |
RESULTS |
Exposure of Cortical Slices to A
Increased PKC and CaMKII
Activation in a Time- and Dose-dependent Manner--
To
identify possible early cellular responses to A
treatment that
precede cell death, we examined the effect of A
peptides on the
activation of two important serine/threonine kinases, PKC and CaMKII,
in cortical slices. Because the catalytic competence of many PKC
isozymes depends on autophosphorylation at the carboxyl terminus on a
conserved residue (32), a phosphospecific pan PKC antibody that detects
PKC isoforms only when phosphorylated at this residue was used to
detect activated PKC. CaMKII is autophosphorylated at
Thr286 when the enzyme is activated in the presence of
Ca2+/calmodulin, leading to the appearance of a sustained
Ca2+-independent activity (33, 34); thus an
anti-Thr286-phosphorylated CaMKII antibody was used to
detect activated CaMKII.
A
25-35, which represents the biologically active region
of A
(35-37), was aged to produce aggregated
A
25-35. Application of aged A
25-35 (10 µM) to cortical slices induced a marked increase in the
activated PKC and CaMKII. The dose dependence of
A
25-35-induced PKC and CaMKII activation is shown in Fig. 1 (A and B). A
small effect could be detected following a 10-min exposure to low
concentrations of aged A
25-35 (0.1-1 µM), and a saturating effect was seen at 10 µM of A
25-35. The total PKC and CaMKII
levels exhibited no change with the A
25-35 treatment
(Fig. 1A). Quantification data exhibited a 5.2 ± 1.2-fold increase of PKC (n = 12, p < 0.001, ANOVA) and a 6.2 ± 1.2-fold increase of CaMKII
(n = 12, p < 0.001, ANOVA) following a
10-min exposure to aged A
25-35 (10 µM;
Fig. 1B). Similarly, the full-length fibrillar A
peptide,
A
1-42, also induced a potent increase in the activated
PKC and CaMKII. The dose dependence of A
1-42-induced
PKC and CaMKII activation is shown in Fig. 1C. A small
effect was detectable following a 10-min exposure to 0.1 µM of aged A
1-42, and a saturating
effect was observed at 1 µM of A
1-42.
Quantification data exhibited a 5.0 ± 1.0-fold increase of PKC
(n = 4, p < 0.001, ANOVA) and a
6.1 ± 1.1-fold increase of CaMKII (n = 4, p < 0.001, ANOVA) following a 10-min exposure to aged
A
1-42 (1 µM; Fig. 1C). A
representative example is shown in Fig. 1C
(inset). These results suggest that A
1-42 is
more potent than A
25-35 in activating PKC and
CaMKII.

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Fig. 1.
Activation of PKC and CaMKII by fibril
A : potency and kinetics. A,
dose dependence of the A 25-35-induced phosphorylation
of PKC and CaMKII. The slices were treated with A 25-35
for 10 min at the indicated concentrations. Extracts of the slices were
immunoblotted with an anti-phospho-PKC or an anti-phospho-CaMKII
antibody, followed by reblotting with antibodies recognizing the total
PKC or CaMKII. B, quantitation of PKC and CaMKII
phosphorylation induced by different concentrations of
A 25-35. C, quantitation from four
experiments showing the dose dependence of the
A 1-42-induced phosphorylation of PKC and CaMKII.
Inset, representative immunoblots from one experiment.
D, time course of the A 25-35-induced
phosphorylation of PKC and CaMKII. The brain slices were incubated in
the absence or presence of aged A 25-35 (10 µM) for the indicated times, and phospho-PKC and
phospho-CaMKII were detected by immunoblotting of slice extracts.
E, quantitation of PKC and CaMKII phosphorylation induced by
A 25-35 treatment for different lengths of time.
F, quantitation from three experiments showing the time
course of the A 1-42-induced phosphorylation of PKC and
CaMKII. Inset, representative immunoblots from one
experiment. *, p < 0.001; #, p < 0.01, ANOVA, compared with control. ctl, control.
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The kinetics of A
-induced activation of PKC and CaMKII was also
tested. As demonstrated in Fig. 1 (D-F), The PKC and CaMKII activation induced by A
25-35 (10 µM) or
A
1-42 (1 µM) showed rapid and transient
kinetics, reaching a peak at 10 min and declining to basal levels
within 30-60 min. In contrast to the potent activation of PKC and
CaMKII by A
25-35 in cortical slices,
A
25-35 failed to elicit the effect in striatal slices
(n = 5; data not shown). Given such a time and dose
dependence of the A
effect on PKC and CaMKII activation, the
following experiments were performed with a 10-min treatment of
A
25-35 (10 µM) or A
1-42
(1 µM) in frontal cortical slices unless otherwise stated.
We then compared the effect of different A
peptides on PKC and
CaMKII activation. As shown in Fig. 2
(A and B), the 10-min treatment with unaged
A
25-35 peptide (10 µM) also induced a
significant increase in activated PKC (2.9 ± 0.55-fold,
n = 6, p < 0.01, ANOVA) and CaMKII
(3.8 ± 0.78-fold, n = 6, p < 0.01, ANOVA), but to a lesser extent than aged A
25-35
(10 µM) (PKC, 5.4 ± 1.3-fold; CaMKII, 6.8 ± 1.5-fold; n = 10, p < 0.001, ANOVA).
To confirm the specificity of the action of A
25-35, its
control peptide containing the reverse sequence A
35-25 was used to treat cortical slices. As shown in Fig. 2 (A and
B), the aged control peptide A
35-25 (10 µM) failed to induce the activation of PKC and CaMKII
(PKC, 1.1 ± 0.16-fold; CaMKII, 1.05 ± 0.17-fold;
n = 6, p > 0.05, ANOVA).

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Fig. 2.
Activation of PKC and CaMKII by different
A peptides. A, immunoblots of
phospho-PKC and phospho-CaMKII. The brain slices were incubated in the
absence or presence of aged A 25-35 (10 µM), unaged A 25-35 (10 µM),
or the aged control peptide A 35-25 (10 µM) for 10 min. Extracts of the slices were immunoblotted
with an anti-phospho-PKC or an anti-phospho-CaMKII antibody, followed
by reblotting with antibodies recognizing the total PKC or CaMKII.
B, quantitation of PKC and CaMKII phosphorylation induced by
different A peptides. C, quantitation from five
experiments showing the activation of PKC and CaMKII by the full-length
fibrillar A 1-42 (1 µM) or
A 1-40 (1 µM) but not by the control
peptide A 40-1 (1 µM). Inset,
representative immunoblots from one experiment. D,
quantitation from four experiments showing PKC and CaMKII
phosphorylation induced by unaged A 1-42 and aged
A 1-42 at different concentrations. Inset,
representative immunoblots from one experiment. *, p < 0.001; #, p < 0.01, ANOVA, compared with control.
ctl, control.
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As shown in Fig. 2C, the 10-min treatment with aged
full-length A
1-42 peptide (1 µM) or
A
1-40 peptide (1 µM), but not the control
peptide A
40-1 (1 µM), also induced a
significant increase in activated PKC and CaMKII. To further determine
the effects of soluble and fibrillar A
1-42, we compared
the potency of unaged and aged A
1-42 on PKC and CaMKII
activation. Soluable A
1-42 elicited an observable effect at the concentration of 0.1 µM, whereas fibrillar
A
1-42 as low as 0.01 µM produced a
detectable effect (Fig. 2D). These data suggest that both
soluable A
1-42 (in the form of monomers and oligomers)
and highly insoluble A
1-42 fibrils are able to activate
PKC and CaMKII, and the fibrillar form of A
1-42 is more
potent (~10-fold) than the soluable form.
A
Induction of PKC and CaMKII Activation Depends on Excitatory
Synaptic Transmission and Ca2+ Influx--
To reveal the
potential mechanisms underlying A
-induced activation of PKC and
CaMKII, we tested the capability of different channel antagonists to
block the A
signaling. As shown in Fig. 3 (A and B), the
effect of A
25-35 (10 µM) on PKC and
CaMKII activation was largely reduced by the NMDA receptor antagonist MK-801 (10 µM; PKC, 2.0 ± 0.5-fold; CaMKII,
2.3 ± 0.6-fold; n = 6). The non-NMDA glutamate
receptor antagonist CNQX (10 µM) also significantly
attenuated the A
25-35 effect (PKC, 1.6 ± 0.4-fold; CaMKII, 1.8 ± 0.43-fold; n = 6).
Blocking L-type voltage-dependent Ca2+ channels
(VDCCs) with nifedipine (10 µM) partially decreased the
A
25-35 effect (PKC, 3.1 ± 0.7-fold; CaMKII,
3.5 ± 0.8-fold; n = 6). Combined application of
all three channel antagonists completely eliminated the
A
25-35 effect (PKC, 1.1 ± 0.2-fold; CaMKII,
1.02 ± 0.21-fold; n = 5, p > 0.05, ANOVA). Shown in Fig. 3 (C and D) are the
effects of A
25-35 on PKC and CaMKII activation in the
presence of TTX or EGTA. Blocking sodium channels and action
potentials with TTX (0.5 µM) abolished the
A
25-35 effect (PKC, 1.05 ± 0.21-fold; CaMKII,
1.02 ± 0.25-fold; n = 6, p > 0.05, ANOVA). Treating cortical slices with the Ca2+
chelator EGTA (2 µM) prevented the A
25-35
activation of PKC and CaMKII (PKC, 1.3 ± 0.28-fold; CaMKII,
1.1 ± 0.23-fold; n = 6, p > 0.05, ANOVA). Similarly, the effect of A
1-42 (1 µM) on PKC and CaMKII activation was almost abolished in
the presence of CNQX or EGTA. Quantification data and a representative example are shown in Fig. 3E. These results suggest that
A
-induced activation of PKC and CaMKII is dependent on the elevated
excitatory activity in glutamatergic synaptic networks and the ensuing
calcium entry through glutamate receptor channels and VDCCs.

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Fig. 3.
A -induced activation
of PKC and CaMKII: the role of excitatory transmission.
A, immunoblots of phospho-PKC and phospho-CaMKII. The brain
slices were preincubated in the absence or presence of various
antagonists for 30 min, followed by incubation with or without
A 25-35 (10 µM) for 10 min. PKC and CaMKII
phosphorylation induced by A 25-35 was reduced in the
presence of the NMDA receptor antagonist MK-801 (10 µM),
the non-NMDA receptor antagonist CNQX (10 µM), or the
L-type VDCC antagonist nifedipine (nif, 10 µM) and was abolished in the presence of MK-801 + CNQX + nifedipine. B, quantitation of
A 25-35-induced fold increase of PKC and CaMKII
phosphorylation. *, p < 0.001; #, p < 0.01, ANOVA, compared with the A 25-35 effect under
control conditions ( ). C, immunoblots of phospho-PKC and
phospho-CaMKII. The brain slices were preincubated in the absence or
presence of TTX (0.5 µM) or EGTA (2 µM) for
30 min, followed by incubation with or without A 25-35
(10 µM) for 10 min. PKC and CaMKII phosphorylation
induced by A 25-35 was eliminated in the presence of the
sodium channel blocker TTX or calcium chelator EGTA. D,
quantitation of A 25-35-induced fold increase of PKC and
CaMKII phosphorylation. *, p < 0.001, ANOVA, compared
with the A 25-35 effect under control conditions ( ).
E, quantitation from four experiments showing the blockade
of A 1-42-induced (1 µM) increase of PKC
and CaMKII phosphorylation by CNQX and EGTA. Inset,
representative immunoblots from one experiment. *, p < 0.001; #, p < 0.01, ANOVA, compared with the
A 1-42 effect under control conditions ( ).
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The A
-induced Increase in Activated PKC and CaMKII Is Inhibited
by Activation of M1 Muscarinic Receptors--
We next examined what
can regulate the A
signaling in cortical circuits. Because enhancing
cholinergic transmission is the most effective therapeutic strategy in
AD treatment (26, 27), we first tested the capability of muscarinic
(mAChR) and nicotinic (nAChR) receptor agonists to block the
A
effect. As shown in Fig.
4A, pretreatment of cortical
slices with the mAChR agonist oxo-M (10 µM, 30 min)
almost completely abolished the A
25-35-induced activation of PKC (0.95 ± 0.23-fold, n = 8, p > 0.05, ANOVA) and CaMKII (0.9 ± 0.21-fold,
n = 8, p > 0.05, ANOVA). In contrast, the nAChR agonist nicotine (10 µM, 30 min) did not affect
the A
25-35 activation of PKC (5.0 ± 1.3-fold,
n = 5, p < 0.001, ANOVA) and CaMKII
(6.2 ± 1.5-fold, n = 5, p < 0.001, ANOVA). Similarly, the 5-HT2 serotonin
receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) (10 µM, 30 min) also failed to affect the
A
25-35 activation of PKC (5.5 ± 1.4-fold,
n = 6, p < 0.001, ANOVA) and CaMKII
(6.8 ± 1.6-fold, n = 6, p < 0.001, ANOVA).

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Fig. 4.
Regulation of
A -induced PKC and CaMKII activation: the role
of M1 mAChRs. A and B, quantitation of
A 25-35-induced fold increase of PKC and CaMKII
phosphorylation. The brain slices were preincubated in the absence or
presence of various agents for 30 min, followed by incubation with or
without A 25-35 (10 µM) for 10 min. PKC
and CaMKII phosphorylation induced by A 25-35 was
abolished in the presence of the mAChR agonist oxo-M
(oxo, 10 µM, A) but was unaffected
by the nAChR agonist nicotine (10 µM, A) or
the serotonin (5-HT2) receptor agonist DOI
(10 µM, A). The M1/M4 antagonist pirenzepine
(piz, 10 µM, B), but not the M2
antagonist methoctramine (met, 10 µM,
B), eliminated the capability of oxo-M to inhibit
A 25-35-induced phosphorylation of PKC and CaMKII.
Insets (A and B), representative
immunoblots from single experiments. *, p < 0.001, ANOVA, compared with the A 25-35 effect under control
conditions ( ). C, quantitation showing that oxo-M
inhibited the A 1-42-induced (1 µM)
increase of PKC and CaMKII phosphorylation, and this effect of oxo-M
was blocked by pirenzepine (10 µM) and the highly
selective M1 antagonist MT-7. Different concentrations of MT-7 (0.01, 0.1, and 1 µM) were tested. D, quantitation
showing the effect of oxo-M preincubation for different durations on
A 1-42-induced (1 µM) increase of PKC and
CaMKII phosphorylation. The brain slices were pretreated with oxo-M (10 µM) for different durations (0-60 min), followed by
incubation with A 1-42 (1 µM) for 10 min.
All the treatment with oxo-M blocked the A 1-42
activation of PKC and CaMKII. E, quantitation showing the
recovery kinetics of the A 1-42-induced (1 µM) increase of PKC and CaMKII phosphorylation after
washing off oxo-M. The brain slices were pretreated with oxo-M (10 µM) for 30 min, and then the oxo-M was washed off.
Different lengths of time (0-30 min) elapsed before
A 1-42 (1 µM) was added and incubated for
10 min. About 20 min after washing off oxo-M, A 1-42 was
able to activate PKC and CaMKII. Insets (C-E),
representative immunoblots from single experiments. *,
p < 0.001; #, p < 0.01, ANOVA,
compared with the A 1-42 effect under control conditions
( ). F, histogram summary of the phosphorylation of PKC
substrates in cortical slices treated with or without
A 1-42 in the absence or presence of oxo-M
(n = 4; *, p < 0.001, ANOVA). Cortical
slices that have been pretreated with or without oxo-M (10 µM, 30 min) were incubated with or without aged
A 1-42 (1 µM) for 10 min. Lysates of these
slices were used for immunoprecipitation with anti-PKC. PKC kinase
activity of the immune complex was measured using myelin basic protein
(MBP) as the substrate. Inset, in
vitro kinase activity of PKC immunoprecipitates from one
experiment.
|
|
Among the five subtypes of mAChRs, M1-M5 (38), all types apart from M5
are expressed in cortical neurons (39, 40). We then determined the
potential mAChR subtype(s) involved in the regulation of A
signaling
using specific antagonists. As shown in Fig. 4B, in the
presence of the M1/M4 antagonist pirenzepine (1-10 µM),
oxo-M lost the ability to inhibit A
25-35-induced activation of PKC (5.2 ± 1.3-fold, n = 6, p < 0.001, ANOVA) and CaMKII (6.5 ± 1.45-fold,
n = 6, p < 0.001, ANOVA). On the
contrary, the M2 antagonist methoctramine (10 µM) did not
prevent the oxo-M inhibition of A
25-35 activation of
PKC (0.99 ± 0.28-fold, n = 6, p > 0.05, ANOVA) and CaMKII (0.97 ± 0.31-fold, n = 6, p > 0.05, ANOVA). Similarly, the
A
1-42-induced activation of PKC and CaMKII was also
significantly inhibited by oxo-M, and this effect of oxo-M was blocked
by pirenzepine (Fig. 4C). Given the limited selectivity of
pirenzepine (~5-fold) for M1 and M4 receptors (41, 42), we used MT-7,
a highly selective (>10,000-fold) ligand for M1 receptors (43), to
confirm the involvement of M1 receptors. As shown in Fig.
4C, MT-7 (0.1 µM) eliminated the ability of
oxo-M to inhibit A
1-42 activation of PKC (4.3 ± 1.6-fold, n = 4, p < 0.001, ANOVA) and
CaMKII (5.9 ± 1.7-fold, n = 4, p < 0.001, ANOVA), indicating that the effect of oxo-M is mediated by M1
receptors. These results thus suggest that activation of M1 receptors
can block A
signaling in cortical networks.
To further analyze the inhibition of A
actions by muscarinic
stimulation, we pretreated cortical slices with oxo-M for different durations (0, 10, 30, and 60 min) before adding A
1-42
and incubated for 10 min. As shown in Fig. 4D, no matter how
long the pretreatment with oxo-M was (0-60 min), subsequent addition of A
1-42 failed to induce any further activation of PKC and CaMKII. There was a small activation of PKC and CaMKII during the
initial muscarinic stimulation, but the muscarinic action returned to
base line after 10-30 min. These data suggest that the constant
presence of muscarinic activation prevents the A
-initiated signaling. When we pretreated cortical slices with oxo-M and then washed it out to prevent further muscarinic activation, subsequent addition of A
1-42 regained the ability to activate PKC and CaMKII after 20 min (Fig. 4E).
We also performed in vitro kinase assay to confirm that the
A
1-42 activation of PKC can be blocked by muscarinic
stimulation. As shown in Fig. 4F, application of aged
A
1-42 (1 µM, 10 min) to cortical slices
induced a potent increase of PKC catalytic activity, as measured by the
phosphorylation of its substrate myelin basic protein (5.2 ± 0.9-fold, n = 4, p < 0.001, ANOVA), consistent with the results obtained with the PKC phosphospecific antibody. Pretreatment of cortical slices with oxo-M (10 µM, 30 min) abolished the A
1-42-induced
activation of PKC with the enzymatic assay (0.98 ± 0.1-fold,
n = 4, p > 0.05, ANOVA).
Muscarinic Receptors Block A
Signaling through the Enhancement
of GABAergic Inhibitory Transmission--
What might account for the
muscarinic inhibition of A
signaling? Because the A
activation of
PKC and CaMKII appears to be dependent on the enhanced glutamatergic
excitatory synaptic activity (Fig. 3), muscarinic receptors should
attenuate the A
effect if they can enhance the counteracting
GABAergic inhibitory synaptic activity. We therefore tested the oxo-M
effect on A
signaling when the GABA system is manipulated. As shown
in Fig. 5 (A and B), application of the GABAA receptor enhancer
diazepam (5 µM) exerted a similar effect as oxo-M:
blocking the A
25-35-induced activation of PKC (1.2 ± 0.27-fold, n = 6, p > 0.05, ANOVA)
and CaMKII (1.25 ± 0.29-fold, n = 6, p > 0.05, ANOVA). Moreover, diazepam largely occluded
the effect of subsequent oxo-M application (PKC, 1.02 ± 0.26-fold; CaMKII, 0.98 ± 0.28-fold; n = 5, p > 0.05, ANOVA). To further confirm that the oxo-M
suppression of A
25-35 signaling is dependent on the
muscarinic enhancement of GABAergic inhibition, we blocked
GABAA receptor channels with bicuculline (5 µM). As shown in Fig. 5 (C and D),
in the presence of bicuculline, oxo-M failed to inhibit
A
25-35-induced activation of PKC (5.1 ± 1.4-fold,
n = 6, p < 0.001, ANOVA) and CaMKII
(6.1 ± 1.6-fold, n = 6, p < 0.001, ANOVA). Likewise, diazepam also blocked the A
1-42-induced activation of PKC and CaMKII (Fig.
5E), mimicking the inhibitory effect of oxo-M on
A
1-42 signaling (Fig. 4E). Moreover,
bicuculline prevented the oxo-M inhibition of A
1-42 activation of PKC and CaMKII (Fig. 5E). These data suggest
the requirement of GABAergic inhibitory synaptic transmission in the muscarinic regulation of A
actions.

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Fig. 5.
Muscarinic regulation of
A -induced PKC and CaMKII activation: the role
of inhibitory transmission. A and C,
immunoblots of phospho-PKC and phospho-CaMKII. The brain slices were
preincubated in the absence or presence of various agents for 30 min,
followed by incubation with or without A 25-35 (10 µM) for 10 min. The GABAA receptor enhancer
diazepam (DZ, 5 µM, A) abolished
the A 25-35-induced PKC and CaMKII phosphorylation and
occluded the effect of oxo-M (oxo, 10 µM,
A). In the presence of the GABAA receptor
antagonist bicuculline (bicu, 5 µM,
C), oxo-M failed to inhibit A 25-35-induced
phosphorylation of PKC and CaMKII. B and D,
quantitation of A 25-35-induced fold increase of PKC and
CaMKII phosphorylation. *, p < 0.001, ANOVA, compared
with the A 25-35 effect under control conditions ( ).
E, quantitation from four experiments showing that diazepam
suppressed the A 1-42-induced (1 µM)
increase of PKC and CaMKII phosphorylation, and the inhibitory effect
of oxo-M on A 1-42 activation of PKC and CaMKII was
blocked by bicuculline. Inset, representative immunoblots
from one experiment. *, p < 0.001, ANOVA, compared
with the A 1-42 effect under control conditions
( ).
|
|
To provide more direct evidence for the mechanisms underlying
muscarinic inhibition of A
signaling, we performed
electrophysiological experiments to test the impact of A
and oxo-M
on excitatory and inhibitory transmission in cortical slices. The
synaptic activity of frontal cortical pyramidal neurons in response to
A
exposure was first examined using whole cell current clamp
recordings. As shown in Fig. 6
(A and B), bath application of
A
25-35 (10 µM) or A
1-42
(2 µM) induced excitatory postsynaptic potentials and
triggered sustained action potentials (A
25-35, n = 8; A
1-42, n = 5).
Furthermore, whole cell voltage clamp recordings show that
A
25-35 (10 µM) or A
1-42
(2 µM) caused a marked increase in the frequency and
amplitude of sEPSCs. A representative example is shown in Fig. 6
(C-G). As summarized in Fig. 6H, in a sample of
neurons examined, A
25-35 increased the mean sEPSC
amplitude by 42.9 ± 5.5% (mean ± S.E., n = 8, p < 0.001, K-S test) and the mean sEPSC frequency
by 141.9 ± 39.1% (n = 8, p < 0.001, K-S test). Similarly, A
1-42 increased the mean
sEPSC amplitude by 40.5 ± 10.2% (n = 5, p < 0.001, K-S test) and the mean sEPSC frequency by
131.9 ± 30.1% (n = 5, p < 0.001, K-S test). In contrast to the potent enhancement of sEPSCs,
A
25-35 had no effect on sIPSCs (n = 6, data not shown). On the other hand, oxo-M induced a potent increase in
the frequency and amplitude of sIPSCs. A representative example is
shown in Fig. 7 (A-E). As
summarized in Fig. 7F, in a sample of neurons examined,
oxo-M increased the mean amplitude of sIPSCs by 52.3 ± 9.3%
(mean ± S.E., n = 7, p < 0.001, K-S test) and the mean frequency of sIPSCs by 256.1 ± 48.4%
(n = 7, p < 0.001, K-S test). Taken
together, these results suggest that A
exposure activates excitatory
synapses in cortical circuits, and muscarinic receptors enhance the
counteracting inhibitory transmission, which could lead to the blockade
of the A
signaling.

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Fig. 6.
A 25-35-induced changes in
membrane excitability and excitatory synaptic transmission in frontal
cortical pyramidal neurons. A and B, whole
cell current clamp recordings showing that exposure to aged
A 25-35 (10 µM, A) or
A 1-42 (2 µM, B) induced a
slight depolarization and triggered bursts of action potentials.
Scale bars, 10 mV, 1 min. C-E, representative
sEPSC traces recorded from a voltage-clamped cortical pyramidal neuron
under control condition (C), during bath application of
A 25-35 (D), and after washing off the
peptide (E). Scale bars, 50 pA, 100 ms.
F and G, cumulative plots indicating that the
distribution of sEPSC amplitude (F) and frequency
(G) was reversibly increased by A 25-35 in
the neuron. H, histograms (means ± S.E.) showing the
percentages of increase of sEPSC amplitude (Amp) and
frequency (Freq) by A 25-35
(n = 8) and A 1-42 (n = 5). ctl, control.
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Fig. 7.
Effects of muscarinic receptors on inhibitory
synaptic transmission in frontal cortical pyramidal neurons.
A-C, representative sIPSC traces recorded from a
voltage-clamped cortical pyramidal neuron under control conditions
(A), during bath application of oxo-M (20 µM)
(B), and after washing off the agonist (C).
Scale bars, 100 pA, 100 ms. D and E,
cumulative plots indicating that the distribution of sIPSC amplitude
(D) and frequency (E) was reversibly increased by
oxo-M in the neuron. F, histograms (means ± S.E.)
showing the percentages of increase of sIPSC amplitude (Amp)
and frequency (Freq) by oxo-M (n = 7).
ctl, control.
|
|
 |
DISCUSSION |
A
-induced Signaling in Cortical Neurons--
Since the
discovery of the link between A
deposits and AD, therapeutic
strategies have been aiming at reducing the brain amyloid burden (14,
15). A
is synthesized and secreted by brain cells (44, 45). The
neurotoxicity after prolonged exposure to high concentrations of A
has been established in in vitro models (10, 11). However,
accumulation of diffuse deposits of A
in the brain is an early event
in the development of AD when there is no massive neuronal death,
emphasizing the importance of elucidating the initial neuronal
responses to A
fibrils that precede neurodegeneration. The
significance of understanding the physiological roles of A
apart
from its neurotoxic effects is perhaps more clear in aged APP
transgenic mice (46), where numerous A
plaques are seen in cortical
and limbic structures; yet deficits in synaptic plasticity and spatial
working memory are accompanied by minimal or no loss of presynaptic or
postsynaptic elements (47, 48). Therefore, the A
-induced dysfunction
of cortical and hippocampal neurons, not their death, may be largely
responsible for the impairments in learning and memory associated with
AD.
Previous studies have found that A
administration induced a marked
increase in the tyrosine phosphorylation of multiple proteins in nerve
cell lines and cortical cultures (49-51), suggesting that A
has the
potential to modulate cellular responses to growth factors and
extracellular matrix molecules. In this study, we report a rapid and
potent activation of two important serine/threonine kinases, PKC and
CaMKII, in response to A
25-35, A
1-42, and A
1-40 in cortical slices. The fibrillar A
peptides (A
25-35 and A
1-42) were shown
to be more potent than their soluable forms in eliciting the activation
of PKC and CaMKII, whereas the full-length A
1-42 and
A
1-40 were found to be more potent than
A
25-35 in this process. The A
25-35
activation of PKC and CaMKII was not found in HEK293 cell lines (data
not shown), indicative of the requirement of some neural specific
elements in the A
action. Because PKC and CaMKII activation could
change the phosphorylation state and functional properties of many
downstream targets including neurotransmitter receptors, ion channels,
presynaptic terminal proteins, and cytoskeletal molecules (52-54),
A
might modulate a wide variety of neuronal functions ranging from
synaptic plasticity and transmitter release to neurite outgrowth via
the regulation of PKC and CaMKII.
Mechanisms for A
Signaling--
One of the major mechanisms for
the A
-induced cell death is calcium overload and disruption of
calcium homeostasis (55). In this study, pharmacological evidence
suggests that A
activation of PKC and CaMKII is dependent on
Ca2+ influx resulting from A
-induced excitation of
glutamatergic synapses, consistent with previous calcium imaging
results showing that application of A
25-35 to cultured
hippocampal neurons caused increases in the intracellular
Ca2+ (56). Several agents that interfere with glutamatergic
transmission, including CNQX, MK-801, and TTX, were effective in
eliminating or reducing the A
activation of PKC and CaMKII, whereas
blocking GABAergic transmission with bicuculline was without effect
(Fig. 5, C and D), indicating that the A
action specifically required activation of the excitatory network.
Following A
activation of glutamate synapses, Ca2+ could
enter via several routes, including NMDA receptors, VDCCs, and
Ca2+-permeable AMPA/KA receptors.
Parallel electrophysiological studies in cortical pyramidal neurons
show that application of A
induced excitatory postsynaptic potentials and bursts of action potentials, indicating that A
elevated the excitability of these glutamatergic projection neurons. Neither AMPA- nor NMDA-evoked currents were significantly
affected by A
25-35 (data not shown), suggesting a lack
of effect of A
on postsynaptic glutamate receptor activity. The
A
-induced increase in sEPSC frequency and amplitude confirms that
A
caused an increase in glutamate release in cortical circuits.
Previous studies have shown that A
induces Ca2+ influx
through VDCCs in cortical neurons and nerve cell lines (57, 58),
activates large, nonselective cation currents in sympathetic and
cortical neurons (59, 60), and inhibits several potassium currents in
basal forebrain cholinergic neurons (61). Thus the A
-induced
alteration of cellular ionic activity through interaction with existing
channels or de novo channel formation (62) might be one of
the key mechanisms underlying the A
activation of neuronal excitability.
Regulation of A
Signaling by Muscarinic Receptors--
Given
the strong activation of PKC and CaMKII by fibrillar A
, it is
important to know what can potentially modulate this signaling. Our
data suggest that activation of muscarinic receptors potently
down-regulated the A
action, and this effect was specific for M1
receptors. Furthermore, A
lost the ability to activate PKC and
CaMKII as long as there was constant M1 receptor activation. About 20 min after M1 receptors were inactivated, the ability of A
to
activate PKC and CaMKII recovered. Therefore, activation of muscarinic
receptors will not only modify APP processing and inhibit A
production (22, 23, 63) but also suppress A
functioning (the present
study). The cholinergic deficiency in AD brains could lead to the loss
of this negative regulation of amyloidogenic A
, which in turn could
impair M1 signal transduction (64, 65) and inhibit ACh synthesis and
release (66, 67), aggravating further the cholinergic hypofunction
(21). Because these "vicious cycles" could potentially be inhibited
by M1 agonists, it suggests that enhancing M1 signaling is a promising
point of pharmacological intervention in the treatment of AD (68,
69).
Mechanisms for Muscarinic Regulation of A
Signaling--
Previous studies have shown that activation of
muscarinic receptors increases the excitability of cortical GABAergic
interneurons (70, 71). Because the A
activation of PKC and CaMKII
was dependent on enhanced excitatory glutamatergic transmission, we postulated that enhancing the GABAergic inhibitory synaptic activity may account for the muscarinic inhibition of A
signaling. Both pharmacological and electrophysiological evidence supports this hypothesis. Several manipulations that interfere with GABA
transmission, such as enhancing postsynaptic responses to GABA with
diazepam or blocking GABAA receptor functions with
bicuculline, were effective in mimicking or blocking the muscarinic
inhibition of A
signaling, respectively. Moreover, muscarinic
agonists potently increased inhibitory postsynaptic currents recorded
in pyramidal neurons of frontal cortex. By potentiating GABA inhibition
to counteract the elevated glutamate excitation elicited by A
exposure, muscarinic receptors not only abrogated the A
activation
of PKC and CaMKII but also can potentially suppress the A
-induced
susceptibility to excitatoxicity in cortical circuits.
 |
ACKNOWLEDGEMENT |
We thank Xiaoqing Chen for technical support.
 |
FOOTNOTES |
*
This work was supported by National Science Foundation Grant
IBN-0117026 (to Z. Y.), National Institutes of Health Grant
MH63128 (to Z. Y.), and Howard Hughes Medical Institute Biomedical
Research Support Program Grant 53000261 (to SUNY at Buffalo).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 Physiology
and Biophysics, State University of New York at Buffalo, 124 Sherman
Hall, Buffalo, NY 14214. E-mail: zhenyan@buffalo.edu.
Published, JBC Papers in Press, February 26, 2003, DOI 10.1074/jbc.M209892200
 |
ABBREVIATIONS |
The abbreviations used are:
A
,
-amyloid
peptide;
AD, Alzheimer's disease;
APP,
-amyloid precursor protein;
PKC, protein kinase C;
CaMKII, Ca2+/calmodulin-dependent kinase II;
GABA,
-aminobutyric acid;
ANOVA, analysis of variance;
oxo-M, oxotremorine
methiodide;
MK-801, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]
cyclohepten-5,10-imine maleate;
CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione;
MT-7, muscarinic toxin 7;
sEPSC, spontaneous excitatory postsynaptic current;
sIPSC, spontaneous
inhibitory postsynaptic current;
NMDA, N-methyl-D-aspartate;
TTX, tetrodotoxin;
AMPA,
-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid;
KA, kainic
acid/kainate;
K-S, Kolmogorov-Smirnov;
VDCC, voltage-dependent Ca2+ channel;
mAChR, muscarinic cholinergic receptor;
nAChR, nicotinic cholinergic
receptor.
 |
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