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The Journal of Neuroscience, November 15, 1998, 18(22):9386-9393
Novel Domain-Specific Actions of Amyloid Precursor Protein on
Developing Synapses
Takako
Morimoto,
Ikuroh
Ohsawa,
Chizuko
Takamura,
Mariko
Ishiguro,
Yasuko
Nakamura, and
Shinichi
Kohsaka
Department of Neurochemistry, National Institute of Neuroscience,
Kodaira, Tokyo 187-8502, Japan
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ABSTRACT |
The effect of the secretory form of amyloid precursor protein
(sAPP) on synaptic transmission was examined by using developing neuromuscular synapses in Xenopus cell cultures. The
frequency of spontaneous postsynaptic currents (SSCs) was reduced by
the addition of sAPP, whereas the amplitude of impulse-evoked
postsynaptic currents (ESCs) was increased by sAPP. These opposing
effects on spontaneous versus evoked release were separated by using
the specific domain of APP. The C-terminal fragment of sAPP (CAPP) only
reduced SSC frequency and did not affect ESCs. By contrast, the
N-terminal fragment of sAPP (NAPP) did not affect SSC frequency but did
increase ESC amplitude. The reduction of SSC frequency by sAPP appears
to be mediated by activation of potassium channels through a
cGMP-dependent pathway, whereas the increase of ESC amplitude is
mediated by a different pathway involving activation of protein
kinase(s). These results suggest the potential role of sAPP as a
modulator of synaptic activity by two specific domains.
Key words:
amyloid precursor protein; spontaneous synaptic currents; evoked synaptic currents; domain-specific; modulator; synaptic
activity; developing synapses
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INTRODUCTION |
Amyloid precursor protein (APP) is
the source of  A4 peptide, which is a major component of the amyloid
deposits characterized in Alzheimer's disease (AD) (Selkoe, 1991 ).
Although it has not yet been established, there are increasing data
indicating that APP is causally involved in the pathogenesis of AD
(Selkoe, 1991; Mattson et al., 1993). For example, mutations in APP
have been shown to result in the inherited forms of AD in several cases (Hardy, 1992). Moreover, the physiological role of APP in the development and modulation of synaptic functions has been emphasized recently. APP is widely expressed in neuronal cells, even in the developing nervous systems (Card et al., 1988; Kang and Muller-Hill, 1990; Moya et al., 1994; Salbaum and Ruddle, 1994; Yamazaki et al.,
1995) and is accumulated in the synaptic sites (Schubert et al., 1991).
The secretory form of APP (sAPP) is released from membrane-spanning APP
by electrical stimulation of neural circuits in hippocampal slices
(Nitsch et al., 1993), and sAPP has been shown to stabilize
intracellular calcium concentrations in studies on cultured hippocampal
cells (Mattson et al., 1993). This effect of sAPP is attributable to
the activation of potassium channels through a cGMP-dependent pathway
(Furukawa et al., 1996a). Furthermore, modulatory effects of sAPP on
synaptic activities have been reported, particularly in the hippocampal
slices (Ishida et al., 1997). In this report, sAPP lowered the tetanus
frequency to induce long-term depression (LTD) and enhanced long-term
potentiation (LTP) induced by high-frequency stimulation. These results
suggest involvement of APP in neural activation, but whether APPs
enhance or suppress the level of neural activation is still
controversial. Here, we examined the effects of sAPP on the synaptic
activity of a developing neuromuscular synapse in Xenopus
cell cultures. We found that sAPP modulated the synaptic activity by
two specific domains. The C-terminal fragment of sAPP (CAPP) reduced
the frequency of the spontaneous synaptic currents (SSCs), and did not
affect the evoked synaptic currents (ESCs). By contrast, the N-terminal
fragment of sAPP (NAPP) did not affect SSCs but did increase ESC
amplitude. The reduction of SSC frequency by sAPP appears to be
mediated by activation of potassium channels through a cGMP-dependent
pathway, whereas the increase of ESC amplitude is mediated by a
different pathway involving activation of protein kinase(s). These
results provide direct evidence of the acute domain-specific actions of sAPP in modulating the basal synaptic function of developing synapses.
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MATERIALS AND METHODS |
Preparation of sAPP, N-terminal fragment of APP, and
C-terminal fragment of APP. sAPP, NAPP, and CAPP (Fig.
1) were prepared as described previously
(Ohsawa et al., 1995, 1997). In brief, human APP695 cDNA was subcloned
into pAE1 vector, and NAPP cDNA encoding from Arg16
to Val290 of human APP695 was subcloned into pAE3
vector. CAPP cDNA encoding from Glu305 to
Thr588 of human APP695 was also subcloned. Culture
medium was separated from the yeast transfected with these vectors, and
the proteins were purified using a heparin Sepharose column, an
alkyl-Superose column, and a DE52 column, respectively. The
purification of sAPP, CAPP, and NAPP was monitored by SDS-PAGE and
analyzed by silver staining or Western blotting with monoclonal
antibody 22C11 and Alz-90 (Boehringer Mannheim, Tokyo, Japan). NAPP
contained cysteine-rich motifs and an acidic region, which gave a
higher charge than that of CAPP. Predicted isoelectric points for sAPP,
NAPP, and CAPP were 4.5, 4.0, and 5.5, respectively.
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Figure 1.
Structure of APP, sAPP, NAPP, and CAPP. The
numbers represent the amino acid sequences. The position
of the transmembrane domain (TM) and the
Knitz-type protease inhibitor domain (KPI) are
indicated.
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Cell culture. Xenopus spinal motoneurons and
myotomal myocytes were prepared from stage 20-22 embryos and cultured
according to previously reported methods (Spitzer and Lamborghini,
1976). The dissociated cells were plated on coverslips in the culture medium consisting of 50% (v/v) Leibovitz's medium (Life Technologies, Gaithersburg, MD), 1% (v/v) fetal calf serum (Life Technologies), and
49% (v/v) Ringer's solution (115 mM NaCl, 2 mM CaCl2, 2.5 mM KCl, and 10 mM HEPES, pH 7.3). The cultures were maintained for 1 d at room temperature (24°C) and used for the electrophysiological experiments.
Electrophysiology. Gigaohm-seal whole-cell recording from
muscle cells followed those described previously (Hamill et al., 1981).
All recordings were performed at room temperature (24°C). The
solution inside the whole-cell recording pipette contained 150 mM KCl, 1 mM NaCl, 1 mM
MgCl2, and 10 mM HEPES, pH 7.2. The membrane currents were monitored with a patch-clamp amplifier (Nihon
Koden, Tokyo, Japan). The recorded currents were filtered at 5 Hz,
stored on a DAT recording tape for later playback, and analyzed with
the SCAN program (kindly provided by Dr. J. Dempster, Strathclyde
University, Glascow, UK). Chemicals tested were dissolved in the
culture medium. SSCs were recorded, and the frequency of the SSCs was
determined by counting the number of events per minute. We have
followed the procedure described previously to analyze fluctuations of
ESC amplitude, assuming that evoked acetylcholine (ACh) secretion at
these synapses is quantal in nature (Bekkers and Stevens, 1990; Malinow
and Tsien, 1990; Lo and Poo, 1994). The mean ESC amplitude
(m) and its variance (v) were measured before and
after the addition of sAPP. The distribution of ESC amplitude was
assumed to be a binomial distribution modified to take into account the
variability of the quantal size. Thus, m = aNp and v = a2Np(1  p) + a2Npcm2,
where a is the average quantal size, N is the
total number of available quanta, p is the release
probability, and cm is the coefficient of
variation of the quanta. Coefficient of variation (CV) is defined as
v1/2/m. The square of CV,
CV2 = (1 + cm2 p)/(Np), is a function independent of quantal
size a. If an increase of ESC amplitude by sAPP is
attributable simply to a change in postsynaptic sensitivity, then
CV2 will not change after the addition of sAPP, and
the ratio of CV2 before and after the addition of
sAPP should remain 1. Substantial deviation from this prediction
suggests involvement of presynaptic modulation. Other statistical
analysis was done by ANOVA.
Intracellular calcium monitoring. The intracellular calcium
concentration ([Ca2+]i) was
monitored with the calcium indicator, fura-2 AM (Dojindo, Kumamoto,
Japan) by the modified method described previously (Morimoto et al.,
1998a). In brief, Xenopus neuromuscular cultures were incubated with Ringer's solution supplemented with 10 µM
fura-2 AM for 1 hr at room temperature. After washing twice with
Ringer's solution, cells were exposed at 340 and 360 nm excitation
beams, and fluorescent images were recorded with a highly sensitive
intensifier target camera (Hamamatsu Photonics, Hamamatsu, Japan).
Experiments were performed at room temperature. After image
acquisition, data were expressed as ratios of fluorescence intensity at
340 and 360 nm and analyzed by Argus-50 (Hamamatsu Photonics).
Chemicals. 8-br-cGMP and Rp-8-br-cGMPs were purchased from
Biomol Research Laboratories (Plymouth Meeting, PA). All other chemicals were of analytical grade.
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RESULTS |
Effect of sAPP on spontaneous synaptic currents
Modulatory effects of sAPP on synaptic transmission were examined
at developing neuromuscular synapses in 1-d-old Xenopus nerve-muscle cultures. In the first set of experiments, SSCs were monitored from innervated myocytes by whole-cell voltage-clamp recording. These currents are caused by spontaneous pulsatile ACh
release from nerve terminals, because they are abolished by bath
application of D-tubocurarine and are unaffected by
tetrodotoxin, which blocks action potentials in neurons (Xie and Poo,
1986). As shown in Figure
2B, bath application of
sAPP (20 nM) reduced the frequency of SSCs within 5 min.
Figure 2C shows the mean frequency during the first 15 min
after the addition of culture medium (Fig. 2C,
cont) or sAPP (5 nM, 20 nM)
normalized to the mean frequency for 5 min before the addition. The
reduction of the SSC frequency was dependent on the dose of sAPP added
to the medium, and at 20 nM sAPP the frequency was
significantly reduced (p < 0.05, ANOVA).
Further analysis of SSC properties was performed to investigate whether
the effect of sAPP was on the presynaptic or postsynaptic mechanisms.
We found that the mean amplitude was not significantly affected by the
addition of sAPP (Fig. 2D). Furthermore, there was no
detectable change in the distribution of SSC amplitude (Fig.
3A), rise time (Fig.
3B), or half-decay time (Fig. 3C) after the
addition of sAPP (p > 0.05, ANOVA). These
results suggest that the ACh sensitivity of the postsynaptic sites has
not been affected by the addition of sAPP. Therefore, it is most likely that the primary action of sAPP on these synapses is a modulation of
the presynaptic ACh secretion mechanism.
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Figure 2.
Effect of sAPP on the SSCs. A, B,
The continuous trace shows the membrane currents
recorded from innervated myocytes before and after application of
culture medium (A) and sAPP (20 nM)
(B). Arrows indicate the point at
which the solution was added. The insets below depict
the average traces of SSCs during indicated times at higher time
resolution. Calibration: fast traces, 35 msec, 0.35 nA; slow traces, 3 min, 0.5 nA. C, The frequency of SSCs during the 15 min
after application of culture medium (cont;
n = 8) and sAPP (5 nM,
n = 5; 20 nM, n = 14) was normalized to the frequency of SSCs for the 5 min before
treatment. The data points represent means ± SEM. Values marked
with an asterisk are significantly different from the
control value (p < 0.05, ANOVA).
D, The mean amplitude of SSCs during the 15 min after
application of culture medium (cont) and sAPP was
normalized to the mean amplitude for the 5 min before treatment. The
data points represent means ± SEM.
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Figure 3.
Absence of any changes in the properties of SSCs.
The amplitude (A), rise time
(B), and half-decay time
(C) distributions of SSCs before ( ) and after
( ) APP treatment were compared. Cumulative frequency refers to the
fraction of total events with values smaller than a given value. The
data points represent means ± SEM (n = 14).
All data points are not significantly different between before and
after APP treatment (p > 0.05, ANOVA).
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Effect of sAPP on evoked synaptic currents
To examine the effect of sAPP on the impulse-evoked synaptic
response, the membrane currents of innervated myocytes were recorded in
response to extracellular suprathreshold stimulation of the neuronal
soma at a frequency lower than 0.1 Hz. In Figure
4A, the neuronal soma
was stimulated at the points marked by a small line before and after
the addition of sAPP solution (at the final bath concentration of 20 nM). The amplitude of ESCs was increased after the addition
of sAPP. Figure 4B shows the time-dependent change of
the mean ESC amplitude normalized to the mean ESC amplitude before
treatment. The mean ESC amplitude was calculated from the amplitude of
ESCs evoked by 5-10 test stimuli. A gradual decline in the mean
amplitude of ESCs was usually observed in the recordings from the
control synapses with culture medium, as reported previously (Lohof et
al., 1993; Lo and Poo, 1994), presumably as a result of slow synaptic
depression caused by the test stimulation. The mean amplitude of ESCs
was increased 5 min after the addition of sAPP to the bath. The
increase in ESC amplitude gradually declined after 15 min, as shown in
Figure 4B. The mean ESC amplitude during the 5-15
min after the addition of sAPP normalized to the mean amplitude before
the addition was 1.20 ± 0.13 (n = 21) (Fig.
5B) and was significantly
different from the value of the control synapses to which culture
medium had been added (0.85 ± 0.06, n = 12, p < 0.05, ANOVA). To determine whether the observed
effect of sAPP on ESC amplitude was attributable solely to a change in quantal size, for example, resulting from a change in sensitivity of
postsynaptic ACh receptors, or modulation of presynaptic release mechanisms, the fluctuation of the ESC amplitude was analyzed. As
described in the previous report (Lo and Poo, 1994), the CV of ESC
amplitudes should theoretically remain unchanged after the addition of
sAPP if the effect of sAPP is caused by a change in quantal size. We
measured the CV of ESC amplitudes before
(CVb) and after
(CVa) the addition of sAPP, and as shown
in Figure 4C, the ratios of CV squared
(CVa2/CVb2)
deviated significantly from unity (broken line) in the 17 experiments in which a sufficient number of ESC events were collected
for the analysis. Thus, the increase in ESC amplitude induced by sAPP is unlikely to be accounted for by a simple increase in quantal size.
An increase in the number of available quanta and the probability of
quantal release are likely to be involved.
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Figure 4.
Effect of sAPP on the ESCs. A, The
continuous trace shows the membrane current recorded
from the innervated myocytes before and after application of sAPP. An
arrow indicates the point at which sAPP was added. ESCs
were elicited at a frequency below 0.1 Hz at the times marked with
short vertical line. The insets below
depict the average ESC events at higher time resolution. Calibration:
fast traces, 40 msec, 1.8 nA; slow traces, 2 min, 1.5 nA.
B, Changes in mean amplitude after application of
culture medium () or sAPP (). The mean amplitude of ESCs evoked
by 5-10 test stimuli observed at 5 min intervals after application of
the medium was normalized to the mean amplitude before application. The
data points represent means ± SEM (culture medium,
n = 3-12; sAPP, n = 7-21). An
arrow indicates the point at which the medium was added.
Value marked with an asterisk is significantly
different from the control value (p < 0.05, ANOVA). C, Analysis of ESC amplitude fluctuations for
sAPP-treated synapses (n = 17). The ratio of CVs
squared before and after the addition of sAPP has been plotted versus
the ratio of the mean ESC amplitude before and after sAPP. Each
point represents data from one experiment. The
broken line represents the theoretical prediction for
pure postsynaptic changes in synaptic responses.
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Figure 5.
Effect of the N- and C-terminal domain of sAPP
(NAPP and CAPP) on SSC frequency and ESC amplitude. A,
Averages of the normalized frequency of SSCs after application of
culture medium (Cont, n = 8),
sAPP (n = 14), NAPP
(n = 13), and CAPP
(n = 18). The data points represent means ± SEM. Values marked with an asterisk are significantly
different from the control value (p < 0.05, ANOVA). B, Averages of the normalized mean amplitudes of
ESCs after application of culture medium (Cont,
n = 12), sAPP (n = 21), NAPP (n = 7), and
CAPP (n = 5). The data points
represent means ± SEM. Values marked with one
asterisk and two asterisks are significantly
different from the control value (*p < 0.05;
**p < 0.01, ANOVA).
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Domain-specific effects of sAPP
Several domains of APP have been shown to exhibit the specific
distinct function (Ninomiya et al., 1993; Furukawa et al., 1996b). To
examine the effective domain for two effects of sAPP, i.e., reduction
of SSC frequency and increase of ESC amplitude, we prepared two sAPP
fragments: NAPP, which includes residue 16-290 of APP695, and CAPP,
which includes residues 305-588 of APP695 (Fig. 1). Because sAPP was
divided at the insertion point of the Knitz-type protease inhibitor
(KPI), each fragment could maintain the secondary structure, which is
possibly important to exert the biological activity. SSCs and ESCs were
examined before and after the addition of either NAPP (40 nM) or CAPP (40 nM) into the bath. The
frequency of the SSCs and the mean amplitude of the ESCs for 15 min
after the addition of the factor were normalized to mean values before
their addition (Fig. 5A,B). No reduction in SSC frequency
was observed as a result of the addition of NAPP (n = 13). Surprisingly, an increase of ESC amplitude was detected by the
addition of NAPP. NAPP increased ESC amplitude to 1.20 ± 0.11-fold (SEM, n = 12) of the value before the
addition of NAPP, and the effect was significant
(p < 0.01, ANOVA). The effect of NAPP was
persistent, and ESC amplitude remains elevated to 1.22 ± 0.31-fold of the value before the addition of NAPP at 25 min after the
addition of NAPP. By contrast, the addition of CAPP significantly
reduced the frequency of the SSCs (Fig. 5A)
(n = 18; p < 0.05, ANOVA), with no
effect on the mean amplitude of SSCs (data not shown). On the other
hand, the mean amplitude of ESCs was not affected by CAPP (Fig.
5B). These results suggest that two distinct effects of sAPP
on synaptic activity, reduction of SSC frequency and increase of ESC
amplitude, could be induced by the different domains of sAPP, i.e., by
the C- and the N-terminal domains, respectively.
Mechanisms underlying the two domain-specific effects of sAPP
It has been reported that sAPP can activate K+
channels through the cGMP-dependent pathway, resulting in a reduction
of the cytosolic concentration of calcium ions
([Ca2+]i) in cultured
hippocampal neurons (Furukawa et al., 1996a). Such modulation of neural
excitability was shown to depend on the C-terminal fragment of sAPP
(Furukawa et al., 1996b). Because cytosolic Ca2+ is
known to influence the frequency of spontaneous quantal secretion (Katz
and Miledi, 1969), the reduction of frequency of SSCs by sAPP may be
mediated by the same mechanism. Thus, we first examined the effect of
sAPP on [Ca2+]i of the cultured cells.
Figure 6A shows the
change of [Ca2+]i induced by sAPP.
[Ca2+]i was reduced by the addition of
sAPP in neurons, but there was no change in
[Ca2+]i in muscle cells. CAPP also
reduced [Ca2+]i in neurons (data not
shown). Next, we have examined the effects of a K+
channel activator and cGMP on the SSC frequency (Fig.
6B) and ESC amplitude (Fig. 6C). As shown
in Figure 6B, the K+ channel
activator diazoxide could mimic the effect of sAPP on SSC frequency.
Bath application of diazoxide at a final concentration of 100 µM significantly reduced SSC frequency (n = 7; p < 0.01, ANOVA). Furthermore, 8-br-cGMP, a
membrane permeable analog of cGMP, mimics the effect of sAPP on SSC
frequency. Bath application of 8-br-cGMP at a final concentration of
600-700 µM significantly reduced SSC frequency
(n = 7; p < 0.05, ANOVA). However,
both diazoxide and 8-br-cGMP did not alter ESC amplitude (Fig.
6C). These results suggested that the cGMP-dependent
mechanism is sufficient in reducing the SSC frequency. That
cGMP-dependent activity is necessary for the action of sAPP on the SSC
frequency is tested further by the use of a specific competitor of
cGMP, Rp-8-br-cGMP (RGS). Addition of RGS (30 µM) by
itself had no effect on either SSC frequency (Fig.
6B) or ESC amplitude (Fig. 6C). However,
the same RGS addition inhibited sAPP-induced reduction of SSC frequency (Fig. 6B), without affecting sAPP-induced increase of
ESC amplitude (Fig. 6C). These results strongly suggested
that reduction of SSC frequency by sAPP or CAPP was caused by a
cGMP-dependent pathway and that the increase of ESC amplitude by sAPP
or NAPP was mediated by the different mechanism.
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Figure 6.
Involvement of K+ channel
activation and the cGMP-dependent pathway in the reduction of SSC
frequency. A, Examples of
[Ca2+]i change in the neuromuscular
culture. Fluorescent ratio excised at 340 and 360 nm was measured. sAPP
(20 nM) was added at the indicated time. Reduction of
[Ca2+]i was detected in neurons.
N, Neural cell; M, muscle cell.
B, Effect on SSC frequency of sAPP (20 nM,
n = 14), diazoxide (100 µM,
n = 7), 8-br-cGMP (500-700 µM,
n = 7), RGS (30 µM,
n = 5), and RGS (30 µM) + sAPP (20 nM) (n = 6). SSC frequency for the 15 min after the addition of the chemicals was normalized to the frequency
before their addition. The data points represent means ± SEM.
Values marked with one asterisk and two
asterisks are significantly reduced compared with the control
synapses, to which culture medium was added (cont,
n = 5) (*p < 0.05;
**p < 0.01, ANOVA). C, Effect on
ESC amplitude of sAPP (20 nM, n = 21),
diazoxide (n = 4), 8-br-cGMP (n = 7), RGS (n = 9), and RGS + sAPP
(n = 7). The data points represent means ± SEM. Diazoxide, 8-br-cGMP, and RGS did not affect ESC amplitude
compared with the control synapses to which culture medium was added
(cont, n = 12). Even in the presence
of RGS, sAPP significantly increased ESC amplitude
(*p < 0.05, compared with the control;
p < 0.07, compared with the value of RGS addition;
ANOVA).
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The mechanism for the increase of ESC amplitude by NAPP was
tested further. We have reported recently that sAPP activates phospholipase C and protein kinase C (PKC) in rat neocortical neurons (Ishiguro et al., 1998). Because activation of PKC is known to
affect synaptic transmission at many synapses (Shapira et al., 1987),
we have tested the effect of NAPP in the presence of staurosporine, a
broad-spectrum inhibitor of protein kinases including PKC. An example
of the experiments is depicted in Figure 7A,B. Staurosporine (at the
final concentration of 100 nM) was added to the bath after
the onset of whole-cell recording, and the mean ESC amplitude was
determined 10 min after the addition of drug. NAPP was then added to
the bath, and the mean ESC amplitude was determined 5 min after the
addition of NAPP. As shown in Figure 7C, treatment of
staurosporine by itself did not affect ESC amplitude in the time scale
of these experiments. In the absence of staurosporine, NAPP
significantly increased ESC amplitude (Fig. 7A,C).
Pretreatment of staurosporine abolished the increase of ESC amplitude
by NAPP (Fig. 7B,C). These results suggest that NAPP
increases the ESC amplitude through the activation of protein
kinase(s). Figure 7D showed the effect of CAPP on SSC
frequency in the presence of 100 nM staurosporine. Even in
the presence of staurosporine, CAPP significantly reduced SSC
frequency, suggesting that the reduction of SSC frequency by CAPP was
not caused by activation of protein kinase(s).
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Figure 7.
Mechanisms of the increase of ESC amplitude by
NAPP. A, B, The continuous trace depicts the membrane
current recorded from the innervated myocytes before and after
application of NAPP in the absence (A) or
presence (B) of staurosporine (sp,
100 nM). Neurons were stimulated at the times indicated
(), 10 min after application of culture medium (A,
CM) and staurosporine (B), and 5 min after application of NAPP. Calibration: 5 min, 400 nA.
C, Average of the mean ESC amplitude at 15 min after the
start of recording, normalized to the value 10 min after the start of
recording. Staurosporine was added at the beginning of recording, and
NAPP was added 10 min after the start of recording, as in
A and B. Culture medium was added instead
of the chemicals in each of the control experiments. NAPP also
increased the ESC amplitude with this protocol [n = 7, *p < 0.05, ANOVA, compared with control
(n = 12)]. Pretreatment with staurosporine
significantly inhibited the effect of NAPP on the increase of ESC
amplitude (n = 3, *p < 0.05, ANOVA). The data points represent means ± SEM. D,
Reduction of SSC frequency by CAPP in the presence of staurosporine.
Staurosporine was added at the beginning of the recording. The
arrow indicates the point at which either culture medium
(, n = 5) or CAPP (, n = 8) was added. SSC frequency was normalized to the frequency for the 5 min at the beginning of the recording and averaged. CAPP significantly
reduced SSC frequency even in the presence of staurosporine
(*p < 0.05, ANOVA). The data points represent
means ± SEM.
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DISCUSSION |
In this study, we observed two distinct and opposite effects
of sAPP on the spontaneous and evoked synaptic activity: a reduction of
SSC frequency and an increase in ESC amplitude. Several lines of
evidence suggested that these effects were predominantly a result of
changes in presynaptic ACh release, instead of changes in postsynaptic
ACh response. First, the distribution of SSC amplitude and the time
course remained the same after the addition of sAPP, indicating no
significant alteration of subsynaptic ACh sensitivity. Second, we
observed an increase in the amplitude of ESCs and a reduction in SSC
frequency at the same time by the addition of sAPP, inconsistent with a
simple change in the postsynaptic sensitivity as an underlying
mechanism. Third, analysis of the fluctuation of ESC amplitude further
supports the involvement of presynaptic effect.
It is quite surprising that those two effects of sAPP are
accounted for by the action of different domains of sAPP and are mediated by the different intracellular mechanisms. The reduction of
SSC frequency by sAPP, whose activity resides in CAPP, was mediated by
a cGMP-dependent pathway. This possibility was indicated by the finding
that an activator of cGMP-dependent pathway, 8-br-cGMP, could mimic the
effect of sAPP on SSC frequency and that an inhibitor of the
cGMP-dependent pathway, RGS, could inhibit it (Fig. 6). These results
are consistent with the previous reports that sAPP lowers
[Ca2+]i by activating potassium
channels through a cGMP-dependent pathway in hippocampus cells and that
the effective domain resides in the C-terminal (Furukawa et al.,
1996a,b). We also found that sAPP reduced
[Ca2+]i in neurons and that lowering
the [Ca2+]i by bath application of
BAPTA-AM is known to reduce the frequency of SSC at Xenopus
developing neuromuscular synapses (Girod et al., 1995).
The activity for increasing the ESC amplitude resides in NAPP.
This effect on ESC amplitude is likely to be mediated by the activation
of the protein kinase(s), because it was inhibited by staurosporine.
That the effect on ESCs is mediated by different intracellular
mechanisms from that on SSCs is further supported by the following
findings. First, drugs that influence the effect of sAPP on SSC
frequency were all ineffective in modifying ESC amplitude. Second, in
the presence of staurosporine, CAPP still reduced the SSC frequency. We
showed previously that phospholipase C and PKC are activated and
growth-associated protein-43 (GAP-43) and myristoylated alanine-rich C
kinase substrate are phosphorylated by sAPP in rat neocortical neurons
(Ishiguro et al., 1998). It has been shown that PKC enhances N- and
L-type calcium channel currents in frog sympathetic neurons (Yang and
Tsien, 1993). Prolonged action potential-evoked calcium influx into the
presynaptic terminal through these calcium channels will lead to
increased transmitter release and higher ESC amplitude. GAP-43 and its
phosphorylation by PKC were also shown to be involved in the mechanisms
of neurotransmitter release (Hens et al., 1995). Further studies are
required to clarify the precise mechanism by which the protein kinase
mediates the sAPP effect on ESC amplitude. Whether the effect of sAPP
on ESC amplitude is caused by a reduction of depression of ESC
amplitude or real potentiation of ESC amplitude remains to be
elucidated. Depression of ESC attributable to repetitive testing
stimulation is well known for developing synapses (Lohof et al., 1993;
Lo and Poo, 1994). It is difficult to distinguish between these two possibilities at the present. In either case, the most likely mechanism
of the increase in ESC amplitude induced by sAPP and NAPP is an
increase in the quantal content, namely, an increase in the number of
quanta released by the presynaptic action potential as a result of an
increased probability of release or an increased number of available
quanta or both.
Development and plasticity in synaptic connections depend on the
modulation of synaptic efficacy by electrical and trophic interactions
between presynaptic and postsynaptic cells. Modulation of synaptic
efficacy by sAPP could also play an important role in the development
of the nervous system. Several lines of evidence support this proposal.
Expression of APP was abundant and regulated during development in the
mammalian embryo. Moya et al. (1994) reported that sAPP increased at
the time of synaptogenesis in the hamster visual system and then
declined when mature connections were established, suggesting that
endogenous sAPP may play an important role in target recognition,
synaptic contact, and synaptic maturation in vivo. The APP
of Xenopus is highly homologous to human APP, and its
expression was increased during the embryonic stages (Okado and
Okamoto, 1992). In fact, significant expression of APP mRNA was
detected by RT-PCR even at 22 stage Xenopus embryo, from
which we prepared the culture (data not shown). So, it is likely that
sAPP was secreted endogenously during embryogenesis in vivo.
In the previous study (Morimoto et al., 1998b), reduced SSC frequency
by overexpression of synaptotagmin in developing spinal neurons
resulted in the increased accumulation of synaptic vesicles near the
plasma membrane, which reflected a more mature state of presynaptic
differentiation. Thus the multiple effects of sAPP on synaptic
transmission observed in the present study may reveal a potential
function of APP in the development of neuromuscular synapses. On the
other hand, it is possible that APP also modulates synaptic activity
even in adult neurons because APP is still highly expressed in the
adult. In fact, it has been proposed that sAPP contributes to
activity-dependent synaptic plasticity such as LTP and LTD and learning
and memory. Antibodies against APP have been demonstrated to inhibit
the acquisition of a passive avoidance response in the rat (Doyle et
al., 1990; Huber et al., 1993). Taken together with the report about an
activity-dependent release of sAPP (Nitsch et al., 1993), the effect of
NAPP on ESC amplitude resulted in prolonged enhancement of ESC
amplitude, suggesting its potential role in LTP.
The intracellular signaling pathways of sAPP have been reported,
suggesting the presence of receptor-like protein(s) for APP (Furukawa
et al., 1996a; Ishiguro et al., 1998). Specific high-affinity binding
sites for sAPP on neuronal cells (Ninomiya et al., 1994) also support
the existence of receptor-like protein(s) for APP. The presence of two
domains in sAPP with different effects on the synaptic properties as
shown in the present study suggests the existence of two receptor-like
molecules for sAPP. There are many examples of a single ligand having
multiple types of receptor and mobilizing various different signals in
the cell. Neurotransmitters, such as glutamate and acetylcholine, have
at least two types of receptor: an ion-channel type and a
G-protein-coupled type (Morita and Katayama, 1984; Sugiyama et al.,
1989). There are two kinds of opioid receptors, and they have opposite
functions (Crain and Schen, 1992). There are also two kinds of
receptors for neurotrophic factors, a low-affinity receptor, p75, and
high-affinity Trk receptors (Lewin and Barde, 1996). Regulation
of the expression of the receptor-like molecules results in the
regulated exhibition of these two effects of sAPP. It is worth noting
that some synapses showed only the effect of sAPP on either SSCs or
ESCs. Because neurons in this culture are not homogeneous, this
may suggest the heterogeneity of those neurons, such as heterogeneous
expression of the receptor-like molecules for APP in each neuron.
Further identification of the receptor-like molecule(s) for APP will
clarify the precise physiological function of APP. In addition to the
receptor-like molecules for APP, regulation of APP processing could
also lead to either enhancement or reduction of synaptic activity. It
has been suggested that APP can be processed either (1) via a
nonamyloidogenic pathway in which sAPP is released into the
extracellular space or (2) via an alternative pathway that generates
A-related peptides. A peptide was shown to modulate cholinergic
synaptic activity. In picomole to nanomole order, A peptide acutely
reduced potassium-evoked ACh secretion by cortical and hippocampal
brain slices (Kar et al., 1996). Thus, the distinct function of APP as
a modulator of synaptic activity might be the key for the switch
between physiological and pathological conditions. Enhancement of
evoked synaptic activity by NAPP may suggest its neuroprotective role
against the reduction of synaptic activity induced by A peptide.
In conclusion, we have found that sAPP has two novel synaptic
modulatory actions, each exerted by a distinct domain in the protein.
We have shown further that these actions are mediated by different
intracellular mechanisms. Taken together, these results provide direct
evidence for a multifunctional role of APP in modulating synaptic
functions and suggest a developmental and neuroprotective role of APP.
| |
FOOTNOTES |
Received June 15, 1998; revised Aug. 24, 1998; accepted Sept. 1, 1998.
This study was supported by a Center of Excellence (COE) grant from the
Science and Technology Agency, Japan, and a grant from the Japanese
Ministry of Health and Welfare.
Correspondence should be addressed to Dr. S. Kohsaka, Department of
Neurochemisty, National Institute of Neuroscience, 4-1-1 Ogawa-higashi,
Kodaira, Tokyo 187-8502, Japan.
| |
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Top
Abstract
Introduction
