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The Journal of Neuroscience, August 1, 2001, 21(15):5693-5702
Exploration of Signal Transduction Pathways in Cerebellar
Long-Term Depression by Kinetic Simulation
Shinya
Kuroda1, 2,
Nicolas
Schweighofer1, and
Mitsuo
Kawato1, 3
1 Kawato Dynamic Brain Project, ERATO, Japan
Science and Technology, Kyoto 619-0288, Japan, 2 Division
of Signal Transduction, Nara Institute of Science and Technology, Ikoma
630-0101, Japan, and 3 ATR, Kyoto 619-0288, Japan
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ABSTRACT |
Because multiple molecular signal transduction pathways regulate
cerebellar long-term depression (LTD), which is thought to be a
possible molecular and cellular basis of cerebellar learning, the
systematic relationship between cerebellar LTD and the currently known
signal transduction pathways remains obscure. To address this issue, we
built a new diagram of signal transduction pathways and developed a
computational model of kinetic simulation for the
phosphorylation of AMPA receptors, known as a key step for expressing cerebellar LTD. The phosphorylation of AMPA receptors in
this model consists of an initial phase and an intermediate phase. We
show that the initial phase is mediated by the activation of linear
cascades of protein kinase C (PKC), whereas the intermediate phase is
mediated by a mitogen-activated protein (MAP) kinase-dependent positive
feedback loop pathway that is responsible for the transition from the
transient phosphorylation of the AMPA receptors to the stable
phosphorylation of the AMPA receptors. These phases are dually
regulated by the PKC and protein phosphatase pathways. Both phases also
require nitric oxide (NO), although NO per se does not show any ability
to induce LTD; this is consistent with a permissive role as reported
experimentally (Lev-Ram et al., 1997 ). Therefore, the kinetic
simulation is a powerful tool for understanding and exploring the
behaviors of complex signal transduction pathways involved in
cerebellar LTD.
Key words:
cerebellar long-term depression; kinetics; simulation; signal transduction; positive feedback loop; phosphorylation
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INTRODUCTION |
Cerebellar long-term depression
(LTD), thought to be a molecular and cellular basis for cerebellar
learning (Ito, 1989; Lisberger, 1998; Kawato, 1999), is a process
involving a decrease in the synaptic strength between parallel fiber
(PF) and Purkinje cells (PCs) induced by the conjunctive activation of
PFs and climbing fiber (CF) (Ito, 1989; Linden and Connor, 1995).
Multiple signal transduction pathways have been shown to be involved in
this process (Linden and Connor, 1993; Fiala et al., 1996; Daniel et
al., 1998). PF is thought to transmit its signal through glutamate
(Kano and Kato, 1987; Linden et al., 1991) and nitric oxide (NO)
(Shibuki and Kimura, 1997; Daniel et al., 1998), and CF is thought to
transmit its signal through corticotropin-releasing factor (CRF)
(Miyata et al., 1999) and Ca2+ influx via
voltage-gated Ca2+ channels (Ekerot and
Kano, 1985; Sakurai, 1990; Crepel and Jaillard, 1991; Linden and
Connor, 1993; Daniel et al., 1998). Both PF- and CF-mediated signals
are transmitted into PCs through multiple signaling pathways, including
mitogen-activated protein (MAP) kinase and
Ca2+, and finally lead to the
phosphorylation of AMPA receptors by protein kinase C (PKC) (Fiala et
al., 1996; Daniel et al., 1998; Matsuda et al., 2000). In addition, NO,
by diffusing into PCs, inhibits protein phosphatase through the cGMP
and soluble guanylnyl cyclase (sGC) pathways, resulting in inhibition
of the dephosphorylation of AMPA receptors (Daniel et al., 1998).
Therefore, the signals from PF and CF dually regulate the
phosphorylation of AMPA receptors through the PKC and protein
phosphatase pathways. The phosphorylation of AMPA receptors has been
shown to be a key step for cerebellar LTD expression through
internalization (Matsuda et al., 2000; Wang and Linden, 2000). However,
because of the complex nature of the signaling pathways underlying the
phosphorylation of AMPA receptors in cerebellar LTD, the systematic
relationship between the synaptic inputs and the output responses
remains obscure.
To understand and explore the behaviors of the complex signal
transduction pathways, it is important to use the computational framework of kinetic simulation. Accordingly, by taking advantage of
the recently developed program GENESIS/kinetikit (Bhalla and Iyengar,
1999), we built a computational simulation model for the
phosphorylation of AMPA receptors in cerebellar LTD on the basis of
kinetic parameters. It was reasonable to incorporate only biochemical
parameters of post-translational biochemical reactions, but not those
for gene expression and protein synthesis, into the simulation, simply
because no kinetic parameters of gene expression and protein synthesis
have thus far been available in cerebellar LTD. The phosphorylation of
AMPA receptors in the kinetic model consisted of the initial and
intermediate phases; the former was mediated by
Ca2+, diacylglycerol (DAG), and
arachidonic acid (AA)-mediated PKC pathway and the latter by the
MAP kinase-mediated positive feedback loop pathway. In addition, NO was
required for both the initial and intermediate phases, consistent with
a permissive role as shown experimentally (Lev-Ram et al.,
1997). Therefore, the kinetic simulation of cerebellar LTD
provided us with a novel method for understanding and exploring the
complex nature of the signal transduction pathways involved in
cerebellar LTD.
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MATERIALS AND METHODS |
Block diagram showing the phosphorylation of AMPA
receptors in cerebellar LTD. The conjunctive stimulation of PF and
CF has been shown to elicit cerebellar LTD (Ito, 1989). In addition, multiple signal transduction pathways have been shown to regulate cerebellar LTD (Ito, 1989; Linden and Connor, 1993; Fiala et al., 1996;
Daniel et al., 1998). According to the literature described below, a
new block diagram of cerebellar LTD was reconstructed (see Fig.
1A). Cerebellar LTD has been shown to be mediated by the modulation of AMPA receptors (Linden and Connor, 1993; Daniel et
al., 1998), and the decrease in the EPSP has been thought to be
mediated by the internalization of phosphorylated AMPA receptors (Matsuda et al., 2000; Wang and Linden, 2000). PF mediates its signals
to PCs by releasing glutamate, resulting in the activation of AMPA
receptors and type-1 G-protein-coupled metabotropic receptors (mGluR1s)
(Aiba et al., 1994; Conquet et al., 1994; Daniel et al., 1998; Ichise
et al., 2000). The activation of AMPA receptors leads to an influx of
Ca2+ through the activation of a
Na+/Ca2+ pump
(Brorson et al., 1994; Daniel et al., 1998) and to the activation of
MAP kinase cascades through the activation of Lyn tyrosine kinase
(Hayashi et al., 1999). The activation of mGluR1 leads to the
activation of GTP-binding proteins, Gq, and the subsequent activation
of phospholipase C (PLC), resulting in the production of DAG and
inositol-1,4,5-phosphate (IP3) (Linden and
Connor, 1993; Fiala et al., 1996; Daniel et al., 1998).
IP3 mobilizes Ca2+
from the internal Ca2+ store through
IP3 receptors (Finch and Augustine, 1998; Takechi et al., 1998). The elevation of Ca2+ leads
to the activation of cytosolic phospholipase A2
(PLA2), resulting in the production of AA and the
subsequent activation of PKC (Bhalla and Iyengar, 1999). PKC has been
shown to be involved in the induction of cerebellar LTD (Crepel and
Krupa, 1988; Linden and Connor, 1991). DAG,
Ca2+, and AA activate protein kinase C
(Nishizuka, 1992).
In addition, PF has recently been shown to produce NO (see Fig.
1B) (Shibuki and Kimura, 1997). NO, diffusing into
PCs, activates sGC, and the activated sGC catalyzes GTP into cGMP
(Bredt and Snyder, 1992; Stone and Marletta, 1996; Lev-Ram et al.,
1997; Daniel et al., 1998). Then, cGMP activates cGMP-dependent protein kinase (PKG) (Wang and Robinson, 1997). PKG phosphorylates its substrate, G-substrate (Nairn et al., 1985; Wang and Robinson, 1997).
Recently, cDNA of G-substrate was cloned (Endo et al., 1999; Hall et
al., 1999), and it was shown that phosphorylated G-substrate
preferentially inhibits protein phosphatase 2A (PP2A) rather than
protein phosphatase 1 (Endo et al., 1999). In this simulation,
therefore, we assumed that the dephosphorylation of AMPA receptors is
mediated by PP2A. CF has been known to depolarize PCs, resulting in the
influx of Ca2+ into PCs through
voltage-gated Ca2+ channels (Ito, 1989;
Linden and Connor, 1993; Daniel et al., 1998). In this study,
therefore, Ca2+ elevation induced by the
stimulation of PF and CF, or PF or CF alone, was assumed on the basis
of the experimental results using Ca2+
indicators (Miyakawa et al., 1992; Midtgaard et al., 1993; Kohda et
al., 1995; Wang et al., 2000), not by the kinetic simulation. Recently,
CRF found in CF was shown to play a permissive role in cerebellar LTD
(Miyata et al., 1999) and to activate MAP kinase without
Ca2+ elevation (Rossant et al., 1999). In
this simulation, therefore, MAP kinase was activated by PKC, Lyn, and
CRF through the activation of Raf and MAP/extracellular
signal-regulated kinase (MEK). Activated MAP kinase
phosphorylates and activates PLA2, resulting in
the production of AA and the subsequent activation of PKC (Bhalla and
Iyengar, 1999). Therefore, the PKC-MAP kinase pathway interacts at
this point. This connection should result in a positive feedback loop
(Bhalla and Iyengar, 1999). In this study, therefore, we attempted to
set the parameters to fit the time course of the phosphorylation of the
AMPA receptors to that of EPSP as reported previously (Chen and
Thompson, 1995). We built the NO/cGMP pathway according to
literature (see Fig. 1B, Table 1). All of the
kinetic parameters in Figure 1B are shown in Table 1.
All of the other kinetic parameters used in this study were based on
previous reports (Bhalla and Iyengar, 1999) and are shown on our web
site
(http://www.erato.atr.co.jp/~kuroda/supplementary_info.html). All of the inputs used in this study are also shown in Appendix 1 (see
Fig. 6).
Kinetic simulations. The phosphorylation of AMPA receptors
in cerebellar LTD was simulated based on the following two biochemical reactions: protein-protein (molecule-molecule) interactions and enzymatic reactions. The protein-protein interactions included interactions such as NO-GS and cGMP-PKG. These reactions can be given
by the following formulation:
Experimentally, in most cases, Kf and
Kb are not available in literature. However,
Kd, the dissociation constant, has
generally been reported. Therefore, based on the reported
Kd values,
Kf and
Kb were calculated by the following
definition:
If A is greatly in excess, the total amount of
product, [AB], that can be formed is limited by the amount
of B. Therefore, the reactions should follow the pseudo
first-order reactions. When the half time
(t1/2) of the pseudo first-order reaction is experimentally obtained, Kf and
Kb can be calculated by the following
equation or by plotting [A] versus apparent rate constant, which can be estimated from t1/2, giving
a line with a y-intercept of Kb and
slope of Kf:
The enzymatic reactions include phosphorylation and
dephosphorylation. These reactions can be given by the following
formulation of Michaelis-Menten:
where E, S, and P denote
enzyme, substrate, and product, respectively.
As in the case of protein-protein interactions, the values of
K1 and
K2 are not generally given in
literature. However, K3 can be
calculated by the experimentally shown
Kcat value, given by
Vmax divided by the concentration of
the enzyme. The values of Km are also
generally reported. Therefore, on the basis of the
Km and K3 values, the
values of K1 and K2 were
calculated by the following definition:
Unless apparent rate constants are available, we assume that
K2 is ~2-20× greater than
K3 because K2 is
generally greater than K3 in many enzymes.
Once the above parameters determined on the basis of the kinetic values
were found to be robust enough to reproduce cerebellar LTD elicited
under normal conditions (Chen and Thompson, 1995), we simulated the
following experiments. All of the numerical computations were performed
with the kinetics library, which is an extension to GENESIS
(Bhalla and Iyengar, 1999).
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RESULTS |
Building a kinetic simulation model for the phosphorylation of AMPA
receptors in cerebellar LTD
To develop a computational kinetics simulation for the
phosphorylation of AMPA receptors in cerebellar LTD, we first developed a block diagram for the phosphorylation of AMPA receptors in cerebellar LTD on the basis of reported data (Fig.
1A). Because recent
evidence has suggested that the phosphorylation of AMPA receptors is a key step for the expression of cerebellar LTD through the
internalization of the phosphorylated AMPA receptors (Matsuda et al.,
2000; Wang and Linden, 2000; Xia et al., 2000), we measured
concentrations of phosphorylated and nonphosphorylated AMPA receptors.
According to literature, cerebellar LTD involves the phosphorylation of AMPA receptors of PCs by both the activation of PKC (Crepel and Krupa,
1988; Linden and Connor, 1993; Fiala et al., 1996; Daniel et al., 1998;
Matsuda et al., 2000) and the inhibition of protein phosphatase by the
NO/cGMP pathway (Nairn et al., 1985; Ito and Karachot, 1992; Ajima and
Ito, 1995; Daniel et al., 1998). Raf, MAP kinase, and PKC have been
shown to form a potential positive feedback loop (Bhalla and Iyengar,
1999) (Fig. 1A, pink and
yellow lines), and PKC has been shown to be regulated by the
Ca2+-, DAG-, and AA-mediated linear
pathway (Fig. 1A, blue and yellow lines) and by the positive feedback loop pathway. Because PP2A has
been shown to be phosphorylated by PKG (Endo et al., 1999), we assume
that the action of the NO/cGMP pathway is finally mediated by PP2A
(Fig. 1A, green line, B, Table
1). Therefore, the phosphorylation of
AMPA receptors is dually regulated by PKC and PP2A. In the block
diagram (Fig. 1A), the number of initial
concentrations of molecules was 28. Of the 28, 22 were determined by
literature (Table 1) or by the kinetic simulation (Bhalla and Iyengar,
1999), and 6 were assumed. The numbers of protein-protein
interactions and enzymatic reactions were 30 and 25, respectively.
Regarding the Kd values, 24 of a total
of 30 reactions were determined by literature or by the kinetic
simulation (Bhalla and Iyengar, 1999), and 6 were assumed. Regarding
the values of Km and
Kcat, 20 of a total of 25 reactions
were determined by literature or by the kinetics simulation (Bhalla and
Iyengar, 1999), and 5 were assumed. The bistable behavior of the
phosphorylation of AMPA receptors in cerebellar LTD (see below) was
robust over a wide range of most of the assumed parameters, but it was
very sensitive to parameters for PKC-Raf and PKC-AMPA receptors
because the former includes parameters for the intersection point of
the positive feedback loop (see below), and the latter includes direct
parameters for the phosphorylation of AMPA receptors. Taking advantage
of the kinetic parameters available in literature with some
assumptions, we built the computational kinetic simulation model of the
phosphorylation of AMPA receptors in cerebellar LTD.
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Figure 1.
A, Block diagram of the
phosphorylation of AMPA receptors in cerebellar LTD. The stimulation of
PF and CF results in the elevation of the phosphorylation of AMPA
receptors through the activation of PKC via the activation of both
linear cascades, including DAG, Ca2+, and AA
(blue and yellow lines), and a positive
feedback loop including PLA2
(pink and yellow
lines), and through the inhibition of protein phosphatase 2A
(green line). The dashed-line box
indicates the generation of Ca2+, which was
reconstituted by the previous observation (Miyakawa et al., 1992;
Midtgaard et al., 1993; Kohda et al., 1995; Wang et al., 2000) but not
by the kinetics simulation. The arrows and
bars denote the stimulatory and inhibitory pathways,
respectively. B, Detailed block diagram of the NO/cGMP
pathway. The bidirectional arrows denote reversible
reactions, and the unidirectional arrows denote
irreversible reactions. Enzymes are located in the middle of the
segment. The numbers indicate the reactions that have
kinetic parameters shown in Table 1.
PDE, Phosphodiesterase.
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The conjunctive stimulation of PF and CF induces the stable
phosphorylation of AMPA receptors in cerebellar LTD
On the basis of the kinetic parameters, we tried to make the
kinetic simulation reproduce the experimental results, i.e., that the
conjunctive stimulation of PF and CF induces cerebellar LTD (Chen and
Thompson, 1995). Cerebellar LTD consists of an initial peak followed by
a stable phase (Chen and Thompson, 1995). It has been shown that gene
expression and protein synthesis are required for the late phase of
cerebellar LTD (Linden, 1996); however, these experimental conditions
are different from those of the kinetic simulation because, under the
experimental conditions, only PCs were used, and the noninvolvement of
NO was assumed (Linden, 1996). By considering the fact that only
post-translational biochemical reactions were incorporated into the
kinetic simulation, it is reasonable to assume that the stable phase of
LTD can be divided into two phases: an intermediate phase maintained by
the post-translational biochemical reactions and a late phase
maintained by the protein synthesis and gene expression. Moreover,
because the time course of the phosphorylation of AMPA receptors in
cerebellar LTD has not been determined and the phosphorylation of AMPA
receptors is important for the expression of cerebellar LTD, we assumed here that the phosphorylation of AMPA receptors also consists of three
phases (initial peak, intermediate phase, and late phase) and built the
simulation to reproduce the time course of EPSP in cerebellar LTD. In
the kinetic simulation, the conjunctive stimulation of PF and CF was
found to induce the phosphorylation of AMPA receptors and to reduce the
concentration of nonphosphorylated AMPA receptors (Fig.
2A). The stimulation of
either PF or CF alone failed to induce the stable phosphorylation of
AMPA receptors in cerebellar LTD (Fig.
3). The time course of the
phosphorylation of AMPA receptors consists of three phases: an initial
phase including the initial peak of the phosphorylated AMPA receptors
(0-10 min), an intermediate phase including the sustained
phosphorylation of AMPA receptors (10-40 min), and a late phase
including the disappearance of the AMPA receptor phosphorylation (>40
min). The time course of the nonphosphorylated AMPA receptors in the initial and intermediate phases is similar to that of the amplitude of
EPSP evoked by the conjunctive stimulation of PF and CF (Chen and
Thompson, 1995) (Fig. 2A). In the late phase,
however, the concentration of the nonphosphorylated AMPA receptors
begins to increase and reaches the basal level at ~90 min because of
the dephosphorylation of the AMPA receptors.
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Figure 2.
Conjunctive stimulation of PF and CF to induce the
stable phosphorylation of AMPA receptors. The simulation was run at 1 Hz for a 5 min conjunctive stimulation of PF and CF. The time course of
the indicated molecule concentration is plotted. A, The
phosphorylation of AMPA receptors induced by the conjunctive
stimulation of PF and CF. The dashed line indicates the
concentration for the phosphorylated AMPA receptors. The dark
line indicates the concentration of the nonphosphorylated AMPA
receptors. The changes of EPSP induced by the 10 min conjunctive
stimulation of PF and CF is replotted ( ) from the earlier
observation (Chen and Thompson, 1995). Note that in the kinetic
simulation, the 5 min stimulation of PF and CF is used because 5 min
stimulation is optimal according to a previous report (Karachot et al.,
1994). B, The activation of PKC by the stimulation. The
dark line indicates the total PKC activity; the
dashed line indicates Ca2+-activated
PKC activity; the dotted line indicates DAG-activated
PKC activity; the dashed-dotted line indicates
AA-activated PKC activity. C, The inhibition of PP2A
activity.
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Figure 3.
Requirement for the conjunctive stimulation of PF
and CF to induce the stable phosphorylation of AMPA receptors. The
simulation was run under conditions similar to those in Figure
2A except that the following simulations were
used: both PF and CF, PF alone, or CF alone. The time courses of the
indicated molecules by the conjunctive stimulation of PF and CF
(straight line), by the stimulation of PF alone
(dotted line), and by the stimulation of CF alone
(dashed line) are plotted. A, The
phosphorylated AMPA receptors; B, active PKC;
C, active PP2A; D, active MAP kinase;
E, active PKG.
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Consistently, the activation of PKC also consists of three phases (Fig.
2B): an initial phase mediated by the direct
activation by Ca2+, DAG, and AA, an
intermediate phase mediated by the activation of the MAP
kinase-mediated positive feedback loop (Fig. 3D), and a late
phase mediated by the inactivation of PKC activity (see also Fig. 5).
This positive feedback loop is essential for the transition from
the transient phosphorylation of AMPA receptors to the stable
phosphorylation of the AMPA receptors and is responsible for the
bistability of the AMPA receptor phosphorylation (Figs. 4, 5).
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Figure 4.
Optimal duration of the stimulation to induce the
stable phosphorylation of AMPA receptors in cerebellar LTD. The
duration of the conjunctive stimulation of PF and CF at 1 Hz was varied
between 0 and 15 min with 1 min intervals, and the simulation was run.
The concentrations of the phosphorylated AMPA receptors
(A), active PKC (B), active
PP2A (C), and active MAP kinase
(D) at 30 min after the onset of the stimulation
are shown.
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Figure 5.
The role of each signaling molecule and
pathway in inducing the phosphorylation of AMPA receptors in cerebellar
LTD. To explore the roles of the signaling molecules and pathways in
inducing the phosphorylation of AMPA receptors in cerebellar LTD, the
simulation was run under the same conditions as in Figure
2A except that the concentration of the indicated
molecules shown below was held at the basal level or the indicated
pathway was deleted. A, The phosphorylated AMPA
receptors; B, active PKC; C, active PP2A;
D, active MAP kinase; E, active PKG.
Blue line, None; yellow line,
PKC; green line, NO; pink line,
Ca2+; cyan line,
PLA2; black line, DAG; brown
line, PLA2 activated by Ca2+
elevation; purple line, PLA2 activated by
MAP kinase. Note that the time courses of the active PP2A
(C) or active PKG (E) were
exactly the same, except without NO (green), and
expressed as a single blue line.
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The PP2A activity markedly decreases by the stimulation and becomes
lowest at 5 min and then gradually reactivates (Fig. 2C). The inactivation of PKC and the reactivation of PP2A lead to the dephosphorylation of the AMPA receptors, resulting in the disappearance of the phosphorylated AMPA receptors in the late phase. The
reactivation of PP2A is mediated by the degradation of NO.
Although NO is degraded very fast and consequently the PKG activity
returns to the basal level after the stimulation (Fig. 3E),
the reactivation of PP2A is very slow because the dephosphorylation
step of G-substrate, an inhibitor of PP2A, is assumed to be quite slow.
Because PP2A activity is inhibited by G-substrate phosphorylated by
PKG, it is interesting to test whether this prolonged phosphorylation of G-substrate really occurs in vivo if antibody that
specifically recognizes the phosphorylated G-substrate will be
available in the future. The inactivation of PKC activity is
mediated by the degradation of PKC itself and by the reactivation of
PP2A activity. Although the reactivation of PP2A is very small, this
small reactivation of PP2A blocks the activation of the positive
feedback loop, and the activity of the loop becomes lower than the
threshold, which is a critical point for bistability, resulting in the
transition from the activation state to the inactivation state of the
loop. These results indicate that the current signal transduction
pathways as shown in Figure 1 are able to produce the initial and
intermediate phases for the phosphorylation of AMPA receptors in
cerebellar LTD elicited by the conjunctive stimulation of PF and CF.
Requirement for the conjunctive stimulation of PF and CF to
induce the stable phosphorylation of AMPA receptors
Cerebellar LTD has been shown to be induced by the conjunctive
stimulation of PF and CF, but not by the stimulation of either PF or CF
alone (Ito, 1989; Daniel et al., 1992; Hemart et al., 1994; Chen and
Thompson, 1995). We therefore asked whether the stable phosphorylation
of AMPA receptors also requires the conjunctive stimulation of both PF
and CF. In the kinetic simulation, the stimulation of PF alone did not
induce the intermediate phase of the phosphorylation of AMPA receptors,
although the initial phase was induced to a lesser extent (Fig. 3,
dotted line). The stimulation of CF did not induce either
the initial or intermediate phase (Fig. 3, broken line). The
difference between the initial phase by the stimulation of PF and that
by the stimulation of CF was caused by the absence of NO production by
the stimulation of CF, but not by that of PF. However, an experimental
stimulation similar to the CF alone stimulation in the kinetic
simulation has been shown to induce an initial sharp decrease in the
EPSC (Daniel et al., 1992; Hemart et al., 1994). The failure of the kinetic simulation to reproduce this observation raises the possibility that other mechanisms in addition to the phosphorylation of AMPA receptors underlie the initial phase of cerebellar LTD (see Discussion).
Optimal duration of the stimulation to induce the stable
phosphorylation of AMPA receptors in cerebellar LTD
We next examined what length of time of the conjunctive
stimulation by PF and CF is required to induce the stable
phosphorylation of AMPA receptors. In the simulation, the stable
phosphorylation of AMPA receptors was not induced by a time <3 min
(Fig. 4A). Time >5 min induced the stable
phosphorylation of AMPA receptors, and the optimal duration of the
stimulation was found to be 6 min. The optimal duration in the
simulation (6 min) of the phosphorylation of AMPA receptors was almost
the same as that of the cerebellar LTD observed in an experiment (5 min) (Karachot et al., 1994). This result indicates that the
phosphorylation of AMPA receptors shows bistability within a middle
time scale of up to 40 min. The activation of PKC also showed a similar
bistability (Fig. 4B). The PP2A activity first
decreased linearly and then reached a plateau (Fig. 4C),
indicating that the inhibition of PP2A does not exhibit bistability.
Therefore, the bistability in the phosphorylation of AMPA receptors is
mediated by the prolonged activation of PKC. The activation of the MAP
kinase-mediated positive feedback loop has been reported to be
responsible for the bistability (Fig. 4D) (Bhalla and
Iyengar, 1999). In addition, we found that the inhibition of PP2A is
required for this bistability (Fig. 5). Because bistability in the
phosphorylation of AMPA receptors has not been shown experimentally,
this result provides an interesting testable prediction of bistability
in the phosphorylation of AMPA receptors in cerebellar LTD. A recently
established antiphosphorylated AMPA receptors antibody (Matsuda et al.,
1999) should allow us to test this prediction in the future.
The role of each signaling molecule and pathway in the induction of
the phosphorylation of AMPA receptor in the cerebellar LTD
We next explored the roles of signaling molecules and pathways in
the phosphorylation of AMPA receptors (Fig. 5). Without PKC activation,
the phosphorylation of AMPA receptors did not increase in the kinetic
simulation (Fig. 5A, yellow line) because of the lack of activation of PKC (Fig. 5B). This finding is
consistent with an earlier observation that the inhibition of PKC
activity results in the complete disruption of LTD in mice expressing
inhibitory peptide of PKC in PCs (De Zeeuw et al., 1998). In contrast,
cerebellar LTD has been shown to be unimpaired in mice lacking the
PKC gene, one of the isoforms of PKC (Chen et al., 1995). This
discrepancy may be attributable to the possibility that PKC isoforms
function redundantly or that there is a PKC-independent pathway.
Various experiments suggest the involvement of PKC in the induction of LTD (Linden and Connor, 1991; Ito and Karachot, 1992). Taken together, the former possibility is more likely. Without NO production, no stable
phosphorylation of AMPA receptors was observed (Fig. 5A,
green line) because of the lack of activation of PKG
(Fig. 5E) and that of inhibition of PP2A (Fig.
5C), despite the fact that PKC was fully activated (Fig.
5B). This finding is consistent with the observation that NO
is essential for the induction of all phases of LTD, although NO itself
shows no ability to elicit LTD (Lev-Ram et al., 1997). In addition,
these results support the idea that LTD is dually regulated by the PKC
and NO pathways. Furthermore, without Ca2+
elevation, the stable phosphorylation of AMPA receptors did not occur
with a lesser initial phase (Fig. 5A, pink
line) because of the partial activation of PKC (Fig.
5B). This finding is also consistent with the fact that
Ca2+ is essential for the induction of all
phases of LTD (Sakurai, 1990; Konnerth et al., 1992). Without DAG
elevation, both the initial and intermediate phases of the AMPA
receptor phosphorylation were observed, but the phosphorylation was
less stable compared with that under the normal condition (Fig.
5A, black line) because of the partial activation
of MAP kinase (Fig. 5D). This finding indicates that DAG
contributes to the stable phosphorylation of AMPA receptors in both the
initial phase and the intermediate phase. Experimentally, it is
difficult to analyze the role of DAG because no specific inhibitors of
DAG are available. Moreover, the inactivation of PLC, which produces
both DAG and IP3, results in the depletion of
both products. Consequently, the simulation is useful for analyzing the
role of signal transduction pathways, which is experimentally difficult
to analyze. Without PLA2 activation, the
intermediate phase of the AMPA receptor phosphorylation disappeared, with a slight reduction in the initial phase (Fig. 5A,
cyan line). The positive feedback loop was
responsible for the intermediate phase because without MAP
kinase-mediated PLA2 activation, the intermediate
phase disappeared without affecting the initial phase (Fig.
5A, purple line) because of the lack of
the stable activation of MAP kinase (Fig. 5D). Lack of the
activation of either Raf or MEK resulted in the same results as that
without MAP kinase-mediated PLA2 activation. This
finding indicates that the positive feedback loop including
PLA2 is responsible for the intermediate phase of
the AMPA receptor phosphorylation. This result is consistent with an
earlier observation that PLA2 regulates the
intermediate phase of LTD (Linden, 1995). Without the activation of
PLA2 by Ca2+
elevation, the initial phase came to be slightly reduced, and the
intermediate phase came to be less stable (Fig. 5A,
brown line).
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DISCUSSION |
The initial phase of the AMPA receptor phosphorylation in
cerebellar LTD was dependent on the direct activation of PKC by linear
cascades including DAG, Ca2+, and AA,
whereas the intermediate phase was mediated by the activation of the
positive feedback loop. The inactivation of PLA2
resulted in the disappearance of the intermediate phase without a
remarkable change in the initial phase, indicating that the positive
feedback loop was responsible for the intermediate phase. Consistent
with this result, the selective inhibition of
PLA2 has been reported to convert cerebellar LTD
to short-term depression (STD), and the application of free unsaturated
fatty acids has been shown to result in an apparent conversion from STD
to LTD in cultured PCs by the stimulation, where STD, not LTD, comes to
be normally induced (Linden, 1995). Taken together, it is likely that
PLA2 and AA serve as regulators of the positive
feedback loop and are responsible for the intermediate phase of LTD.
The lack of the activation of Raf, MEK, or MAP kinase led to the same
result as that without the MAP kinase-mediated
PLA2 activation. MAP kinase and MEK have also
been shown to be required for the induction of both the initial and
intermediate phases of cerebellar LTD (Kawasaki et al., 1999). The
reason that MAP kinase and MEK are not involved in the induction of the
initial phase in the simulation may be attributable to the possibility that an unknown part of the MAP kinase cascade is missing in the simulation. MAP kinase has been shown to be required for the mGlu receptor activity (Kawasaki et al., 1999). Therefore, the
molecular linkage between MAP kinase and mGlu receptors needs to
be clarified and incorporated into the kinetic simulation.
In all phases, the inhibition of PP2A by NO is essential for the
phosphorylation of AMPA receptors. This is consistent with an earlier
observation that NO is essential for cerebellar LTD, but NO itself is
insufficient to induce LTD (Lev-Ram et al., 1997), suggesting that PP2A
acts as a gate signal of cerebellar LTD. This result indicates that a
significant amount of PF stimulation is required to induce cerebellar
LTD via inhibition of PP2A, and that spontaneous PF activity may be
insufficient to inhibit PP2A activity enough to be required for the
stable phosphorylation of AMPA receptors. If only a small amount of
PP2A is easily inhibited by spontaneous PF activity, the PF activity
together with spontaneous CF activity may cause unexpected and
unwarranted cerebellar LTD. Therefore, the reason such a large amount
of PP2A is needed may be caused partially by the role of the NO-PP2A
pathway as a gate signal for the induction of cerebellar LTD. In this
study, spontaneous PF activity was not included. However, if
spontaneous PF activity is included, the PP2A may reach to the some
basal level, which may not be small enough to induce LTD, and should be
inhibited further by NO signal, leading to induced LTD. Therefore, with or without spontaneous PF activity, the action of PP2A would be similar. In addition, PP2A has another role in the kinetic simulation; the dephosphorylation of Raf and MEK. Raf and MEK regulate the positive
feedback loop, and the inhibition of the dephosphorylation of these
molecules was required for the activation of the positive feedback
loop. Therefore, PP2A also has a permissive role in the phosphorylation
of AMPA receptors as well as in the activation of the positive feedback loop.
We compared the phosphorylation of AMPA receptors in the kinetic
simulation and cerebellar LTD shown experimentally. In the initial
phase, some results of the AMPA receptor phosphorylation in the kinetic
simulation did not correlate with the experimental observations of
cerebellar LTD. The stimulation of CF alone did not induce an apparent
initial phase in the kinetic simulation (Fig. 3), whereas a similar
protocol can induce an initial sharp peak of EPSC decrease (Daniel et
al., 1992; Hemart et al., 1994). Additionally, without NO or
Ca2+ elevation, a small peak in the
phosphorylation of the AMPA receptors could still be observed in the
kinetic simulation (Fig. 5A), whereas this initial peak was
not observed by the addition of blockers of
Ca2+ (Sakurai, 1990; Eilers et al., 1997)
or NO (Lev-Ram et al., 1997). These discrepancies raise the possibility
that other mechanisms underlie the expression of the initial phase of
cerebellar LTD. It has been shown that increasing
Ca2+ in dendrite activates
Ca2+-dependent
K+ channels, resulting in shunting
PF-induced EPSP (Gruol et al., 1991; Khodakhah and Ogden, 1993; Muller
et al., 1998). Additionally, it has been shown recently that the
activation of postsynaptic mGluR1 in PC dendrites transiently depresses
synaptic transmissions at PF-PC synapses by presynaptic mechanisms
involving Ca2+ increase in PC dendrites
and retrograde signaling (Levenes et al., 2000) and that retrograde
inhibition of Ca2+ influx occurs through
endogenous cannabinoids at excitatory synapses on PCs (Kreitzer and
Regehr, 2001). These mechanisms are likely to be the main mechanisms
for the expression of the initial phase of cerebellar LTD. The kinetic
parameters in these two mechanisms, although unknown at present, should
allow us to test whether these mechanisms can explain the expression of
the initial phase of cerebellar LTD by use of the kinetic simulation.
Accordingly, the kinetics simulation is a powerful tool for testing
whether the phosphorylation of AMPA receptors can explain the
expression of cerebellar LTD. Lists of the kinetic simulation results,
the corresponding experimental results, and model predictions are summarized in Appendix 2.
The phosphorylation of AMPA receptors has been shown to trigger the
internalization of phosphorylated AMPA receptors (Matsuda et al., 2000;
Wang and Linden, 2000). A recent study has demonstrated that, in
cortical neurons, the activation of AMPA receptors without the
activation of NMDA receptors leads to the rapid and almost complete
internalization of AMPA receptors within 5 min after the stimulation
and the slow reinsertion of the AMPA receptors to the cell surface,
resulting in a reduction in the number of AMPA receptors at the cell
surface (Ehlers, 2000). It is possible that a similar mechanism is
involved in cerebellar LTD. Taken together with our results, it is
likely that the phosphorylation of AMPA receptors is a key step for
cerebellar LTD expression through the internalization of the AMPA
receptors at least in the intermediate phase. However,
the internalization of phosphorylated AMPA receptors was not
incorporated into our model because of the absence of kinetic
parameters. The absence of phosphorylated AMPA receptor
internalization in the kinetic simulation may affect the results
by affecting the balance between the phosphorylated and
nonphosphorylated AMPA receptor concentrations; however, it is likely
that the results would be similar if the internalization step was
incorporated into kinetic simulation because the internalization process itself does not affect the signaling pathways. If the kinetic
parameters of the phosphorylated AMPA receptor internalization is
determined, we can address the question of whether the phosphorylation of AMPA receptors and the subsequent internalization of the AMPA receptors can explain the intermediate phase as well as the initial phase of cerebellar LTD.
There are some discrepancies of the signaling pathways between the
slice and the culture conditions. However, cerebellar LTD can be
elicited robustly under both conditions. For example, under some
experimental conditions (Linden et al., 1991, 1995), NO is not thought
to be essential for the induction of cerebellar LTD. It is possible
that the concentration or the activity of PP2A decreases,
or that an unidentified molecule except NO regulates the PP2A pathways
to compensate and induce LTD under such conditions. Additionally, no
evidence of the roles of PLA2 and AA has been demonstrated in the slice condition. If this positive feedback loop
does not work in the slice, then an alternative feedback loop such as
the Rap1-MAP kinase-mediated positive feedback loop found in PC12
cells (York et al., 1998) is responsible for the intermediate phase.
The fact that cerebellar LTD robustly occurs under both conditions
strongly suggests that LTD is elicited by similar mechanisms in both
conditions even if detailed mechanisms are different. If one pathway
operates in only one of the conditions, changing the concentration of
molecules of a certain type might compensate and induce the cerebellar
LTD, or another redundant pathway having a similar characteristics
might come to be a replacement. Therefore, despite the discrepancies
between these conditions, the kinetic simulation model is still useful
for understanding the mechanism of cerebellar LTD.
It has been shown that glial fibrillary acidic proteins (Shibuki et
al., 1996), expressed in astrocytes but not in neurons, and the  2
subunit of the glutamate receptor channels (Hirano et al., 1995;
Kashiwabuchi et al., 1995) are essential for the induction of LTD. The
phosphorylation of AMPA receptors has been shown to lead to the
internalization of the AMPA receptors (Matsuda et al., 2000; Xia et
al., 2000). Although the detailed mechanisms are still unknown, the
molecules and mechanisms involved should be incorporated into the
kinetics model in the future. Local Ca2+
release within the dendritic spines of PCs has been shown recently to
be required for LTD induction (Miyata et al., 2000). Therefore, the
specific localization of molecules and accessibility should also be
quantitatively determined and incorporated into the kinetics model. In
addition to phosphorylation,
Ca2+-dependent
K+ channels (Gruol et al., 1991; Khodakhah
and Ogden, 1993; Muller et al., 1998), presynaptic retrograde signaling
(Levenes et al., 2000), and retrograde inhibition of presynaptic
Ca2+ influx by endogenous cannabinoids
(Kreitzer and Regehr, 2001) have been shown to underlie the expression
of cerebellar LTD. Electrical processes, such as the channel activity
of AMPA receptors and Ca2+-dependent
K+ channels, coupled with biochemical
processes should also be incorporated into the kinetic simulation,
provided the apparent kinetic parameters come to be determined.
Moreover, the role of Raf in the induction of LTD has yet to be shown
experimentally. Therefore, the present kinetic simulation should not be
regarded as a definitive model, but as a complementary method for
exploring cerebellar LTD in addition to experimental methods. It should
also be emphasized that, even if the simulation can reproduce some
experimental results, this does not exclude the possibility that
unknown pathways or molecules are additionally needed for the induction
of cerebellar LTD. In any case, experiments together with the approach
using kinetic simulation should greatly improve our understanding of the behaviors of the complex biochemical reactions underlying cerebellar LTD.
| |
FOOTNOTES |
Received Feb. 26, 2001; revised May 4, 2001; accepted May 9, 2001.
S.K. was supported by the Inheritance and Variation Group,
PRESTO (Japan Science and Technology Corporation,
Japan). We thank G. Ferriol for his help in the kinetic simulation, M. Ito, T. Hirano, H. Qadota, K. Kaibuchi, and K. Doya for critically
reading this manuscript, and K. Kurihara and N. Matsumoto for their
technical assistance.
Correspondence should be addressed to Shinya Kuroda, Kawato Dynamic
Brain Project, ERATO, Japan Science and Technology, 2-2-2 Hikaridai,
Seika-cho, Souraku-gun, Kyoto 619-0288. E-mail:
kshinya{at}erato.atr.co.jp.
N. Schweighofer's present address: Learning Curve, 2F Fuji Bldg 40, 15-14 Sakuragaoka-cho, Shibuya-ku, Tokyo 150-0031.
| |
APPENDIX 1: The inputs used in this study |
The temporal waveforms of inputs such as
Ca2+, NO and glutamate used in this study
are shown (Fig. 6). The concentration of CRF is assumed to be constantly 0.1 µM during the
stimulation because no data were available. In the case of the
stimulation of PF or CF alone, Ca2+ peak
concentrations are reduced to 2.3 and 2.6%, respectively, according to
an earlier report (Wang et al., 2000).
View larger version (11K):
[in this window]
[in a new window]
|
Figure 6.
The inputs used in this study. The temporal
waveforms of inputs, such as Ca2+
(A), NO (B), and glutamate
(C), which are induced by a single stimulation of
PF and CF, are shown.
|
|
| |
APPENDIX 2: Summary of the kinetic simulation results and the
corresponding experimental results together with the predictions |
The phosphorylation of AMPA receptors and the changes of EPSC (Chen
and Thompson, 1995) induced by the conjunctive stimulation of PF and CF
(Fig. 2)
The initial and the intermediate phases are reproduced by the
kinetic simulation. The late phase is not reproduced because the late
phase of cerebellar LTD has been shown to require gene expression and
protein synthesis (Linden, 1996), and our kinetic simulation does not
include both of them. The time courses of the activation of PKC and
PP2A have yet to be determined. However, antibodies against the
phosphorylated AMPA receptors (Matsuda et al., 2000) and the
phosphorylated G-substrate, an inhibitor of PP2A, should allow us to
measure these time courses in the future.
The conjunctive stimulation of PF and CF induces the stable
phosphorylation of AMPA receptors in cerebellar LTD (Fig. 3)
In the kinetic simulation, neither the initial nor intermediate
phase is significantly induced by the stimulation of either PF or CF
alone. However, an experimental stimulation similar to the CF
alone stimulation in the kinetic simulation has been shown to induce an
initial sharp decrease in the EPSC (Daniel et al., 1992; Hemart et al.,
1994). The failure of the kinetic simulation to reproduce this
observation raises the possibility that other mechanisms in addition to
the phosphorylation of AMPA receptors underlie the initial phase of
cerebellar LTD. It has been shown that increasing
Ca2+ in dendrite activates
Ca2+-dependent
K+ channels, resulting in shunting
PF-induced EPSP (Gruol et al., 1991; Khodakhah and Ogden, 1993; Muller
et al., 1998). Additionally, it has been shown recently that the
activation of postsynaptic mGluR1 in PC dendrites transiently depresses
synaptic transmissions at PF-PC synapses by a presynaptic mechanism
involving Ca2+ increases in PC dendrites
and retrograde signaling (Levenes et al., 2000) and that retrograde
inhibition of Ca2+ influx occurs through
endogenous cannabinoids at excitatory synapse on PCs (Kreitzer and
Regehr, 2001). These mechanisms are likely to be the main mechanisms
for the expression of the initial phase of cerebellar LTD.
Bistability of the phosphorylation of AMPA receptors in cerebellar
LTD (Fig. 4)
In the kinetic simulation, the phosphorylation of AMPA receptors
by PKC shows bistability. Because bistability in the phosphorylation of
AMPA receptors has not been shown experimentally, this result provides
an interesting testable prediction of bistability in the
phosphorylation of AMPA receptors in cerebellar LTD. A recently established antiphosphorylated AMPA receptors antibody (Matsuda et al.,
1999) should allow us to test this prediction in the future.
The role of each signaling molecule and pathway in the induction of
the phosphorylation of AMPA receptor in the cerebellar LTD (Fig. 5)
In the kinetic simulation, the roles of PKC, NO,
Ca2+, and PLA2 are
basically consistent with the experimental observations (Sakurai, 1990;
Konnerth et al., 1992; Lev-Ram et al., 1997; De Zeeuw et al., 1998).
However, without NO or Ca2+ elevation, a
small peak in the phosphorylation of the AMPA receptors can still be
observed in the kinetic simulation, whereas this initial peak is not
observed by the addition of blockers of
Ca2+ (Sakurai, 1990; Eilers et al., 1997)
or NO (Lev-Ram et al., 1997). As indicated above, these discrepancies
raise the possibility that other mechanisms underlie the expression of
the initial phase of cerebellar LTD.
In addition, DAG contributes to the stable phosphorylation of AMPA
receptors in both the initial and intermediate phases in the kinetic
simulation. Experimentally, it is difficult to analyze the role of DAG
because no specific inhibitors of DAG are available. Moreover, the
inactivation of PLC, which produces both DAG and IP3, results in the depletion of both products.
Consequently, the simulation is useful for analyzing the role of signal
transduction pathways, which is experimentally difficult to analyze.
| |
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ABSTRACT
INTRODUCTION
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