Translational switch for long-term maintenance of synaptic plasticity.

Abstract

Memory can last a lifetime, yet synaptic contacts that contribute to the storage of memory are composed of proteins that have much shorter lifetimes. A physiological model of memory formation, long-term potentiation (LTP), has a late protein-synthesis-dependent phase (L-LTP) that can last for many hours in slices or even for days in vivo. Could the activity-dependent synthesis of new proteins account for the persistence of L-LTP and memory? Here, we examine the proposal that a self-sustaining regulation of translation can form a bistable switch that can persistently regulate the on-site synthesis of plasticity-related proteins. We show that an alpha CaMKII-CPEB1 molecular pair can operate as a bistable switch. Our results imply that L-LTP should produce an increase in the total amount of alpha CaMKII at potentiated synapses. This study also proposes an explanation for why the application of protein synthesis and alphaCaMKII inhibitors at the induction and maintenance phases of L-LTP result in very different outcomes.

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Translational switch for long-term maintenance
of synaptic plasticity
Naveed Aslam1, Yoshi Kubota1, David Wells2and Harel Z Shouval1,3,*
1Department of Neurobiology and Anatomy, The University of Texas, Medical School at Houston, Houston, TX, USA,2Department of Cellular, Molecular and
Developmental Biology at Yale University, New Haven, CT, USA and3Department of Biomedical Engineering the University of Texas, Austin, TX USA
* Corresponding author.DepartmentofNeurobiologyandAnatomy,TheUniversityofTexasMedicalSchoolatHouston,6431Fanninstreet.,Houston,TX77030,USA.
Tel.: þ1 713 500 5708; Fax: þ1 713 500 0621; E-mail: harel.shouval@uth.tmc.edu
Received 12.6.08; accepted 13.5.09
Memory can last a lifetime, yet synaptic contacts that contribute to the storage of memory are
composedofproteinsthathavemuchshorterlifetimes.Aphysiologicalmodelofmemoryformation,
long-term potentiation (LTP), has a late protein-synthesis-dependent phase (L-LTP) that can last for
manyhoursinslicesorevenfordaysinvivo.Couldtheactivity-dependentsynthesisofnewproteins
account for the persistence of L-LTP and memory? Here, we examine the proposal that a self-
sustaining regulation of translation can form a bistable switch that can persistently regulate the on-
site synthesis of plasticity-related proteins. We show that an aCaMKII–CPEB1 molecular pair can
operate as a bistable switch. Our results imply that L-LTP should produce an increase in the total
amount of aCaMKII at potentiated synapses. This study also proposes an explanation for why the
application of protein synthesis and aCaMKII inhibitors at the induction and maintenance phases of
L-LTP result in very different outcomes.
Molecular Systems Biology 5: 284; published online 16 June 2009; doi:10.1038/msb.2009.38
Subject Categories: simulation and data analysis; neuroscience
Keywords: bifurcation analysis; protein synthesis; synaptic plasticity
This is an open-access article distributed under the terms of the Creative Commons Attribution Licence,
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permission.
Introduction
Synaptic plasticity, the experience-dependent change in
synaptic efficacies, provides a physiological basis for learning
and memory storage (Bliss and Lomo, 1973; Bliss and
Collingride, 1993; Bear, 1996; Morris et al, 2000). Theoretical
considerationsrequirethatsynapticplasticitymustbesynapse
specific if it is to provide sufficient neuronal selectivity, but
mustalsobelonglastingifitisindeedthebasisforlong-lasting
learning and memory. However, synapses are composed of
proteins that turnover at rates that are much faster than
memory lifetimes (Crick, 1984; Lisman and Goldring, 1988).
Therefore, there must be a synapse-specific biological
mechanism to preserve synaptic efficacies in the face of
protein turnover.
Long-term potentiation (LTP), a long-lasting cellular man-
ifestation of synaptic plasticity, can be divided into two forms:
early-phase LTP (E-LTP) is induced by a ‘weak’ stimulus, is
protein synthesis independent, and lasts for 2–4h, whereas
late-phase LTP (L-LTP), which requires a ‘strong’ stimulus,
lasts much longer and depends on the synthesis of new
proteins (Frey et al, 1988; Kang and Schuman, 1996).
Although both transcription and translation have been
implicated in L-LTP, we concentrate here on the effect of
translation because nuclear transcription cannot account for
synapse specificity.
A popular theory is that the maintenance of long-term
memory and synaptic plasticity is accomplished through a
bistable molecular switch. Specifically, most attention has
been givento theorythat autophosphorylation of aCaMKII can
form a bistable switch that is resistant to phosphatases and
protein turnover (Lisman and Goldring, 1988; Zhabotinsky,
2000; Lisman and Zhabotinsky, 2001; Milleret al, 2005). There
are several significant challenges to this theory, which include
the following observations: First, this theory does not depend
on protein synthesis and cannot account for the protein
synthesis dependence of long-term memory and plasticity
(Frey et al, 1988; Kang and Schuman, 1996); second, this
theory assumes a conserved amount of total aCaMKII and
therefore cannot account for the observed increase in synaptic
levels of aCaMKII (Ouyang et al, 1997, 1999; Otmakhov et al,
2004); third, a key biochemical experiment of aCaMKII
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phosphorylation did not find bistability (Bradshaw et al,
2003). In addition, there is a dispute whether blocking
aCaMKII activity during the maintenance phase reverses
L-LTP (Malinow et al, 1989; Otmakhov et al, 1997; Sanhueza
et al, 2007). Therefore, it makes sense to examine alternative
theories that might be able to account for these experimental
observations.
The main objective of this work is to explore a possible link
between activity-dependent temporal and spatial regulation of
post-transcriptional gene expression and lifelong stability of
some memories, despite the rapid turnover of their molecular
substrates. This study is motivated by the following experi-
mental observations: (1) L-LTP requires new protein synthesis
(Stanton and Sarvey, 1984; Frey et al, 1988). (2) Almost all the
components of translational machinery are constitutively
localized in dendrites (Gardiol et al, 1999; Pierce et al,
2000; Ostroff et al, 2002; Tang et al, 2002). (3) The translation
of proteins important for synaptic plasticity, such as aCaMKII,
can be controlled locally in dendrites and synapses (Wu et al,
1998; Miller et al, 2002). Here, we examine a hypothesis that a
molecular loop between a kinase and a translation regulation
factor acts as a bistable switch to stabilize activity-induced
synaptic plasticityoverlong periods oftime. Variousregions of
the brain, including hippocampus and neocortex, are involved
in different phases of memory consolidation (McClelland et al,
1995; Wiltgen et al, 2004), indicating that the maintenance of
memory includes system-level components that are beyond
cellular and molecular substrates. Discussions on these
system-level components are beyond the scope of this study.
One regulatory mechanism for mRNA translation is
cytoplasmic polyadenylation (Wells et al, 2000, 2001), in
whichthetranslationofmRNAsismodulatedbyregulatingthe
length of the 30poly(A) tail (Wilutz et al, 2001). A shorter
poly(A) tail entails lower translational efficiency (Sheets and
Wickens, 1989). The length of the poly(A) tail is increased
through cytoplasmic polyadenylation, thus enhancing the
efficiency of translation (Gallie, 1991). The translation of
mRNAs that contain a short nucleotide sequence known as
cytoplasmic polyadenylation elements (CPEs) in their 30UTR is
regulatedthrough polyadenylation.
CPE-binding protein (CPEB1), which represses translation
through its dual interactions with CPEs and other mRNA-
binding proteins. The phosphorylation of CPEB1 changes its
interactions with these other proteins, promotes polyadenyla-
tion,andincreasestranslationefficiency(DuandRichter,2005).
A key molecule linked to the induction of LTP is aCaMKII
(Soderling, 2000; Hudmon and Schulman, 2002); however, its
link to the maintenance phase of LTP is less clear (Silva et al,
1992; Bagni et al, 2000). The aCaMKII protein is highly
enriched at synapses and a significant amount of aCaMKII
mRNA is localized in dendrites (Wells et al, 2001). Recent
experimental evidence indicates that almost 83% of aCaMKII
foundindendritesduringnormalbrainfunctionissynthesized
locally in dendrites (Miller et al, 2002). The aCaMKII mRNA
contains twoCPE elements in its 30UTR, andits translationcan
be regulated through a CPEB1-dependent polyadenylation
process. Recent studies (Atkins et al, 2004, 2005) have
shown that aCaMKII can phosphorylate and therefore
possibly modulate its own translation at synapses in a CPE-
dependent manner.
The CPEbindsa
In this study, we examine the hypothesis that a kinase
translation-factor loop acts as a bistable switch and study a
specific instantiation of such a loop, the aCaMKII–CPEB1
molecular pair. Here, using a computational model, we show
that the proposedaCaMKII–CPEB1 molecular loop can act as a
bistable switch and thus stabilize the synaptic efficacies in a
synapse-specific manner despite a continual turnover of its
molecularconstituents.Wealsoshowthatwiththeapplication
of a stimulus pulse (of (Ca2þ)4–CaM), we can induce a long-
lasting elevation of total aCaMKII levels consistent with
experimental observations (Ouyang et al, 1997, 1999; Otma-
khov et al, 2004). The induction of L-LTP also produces a
moderate, persistent elevation in the fraction of phosphory-
lated aCaMKII, in contrast to saturated levels of phosphory-
lated aCaMKII in post-translational models (Lisman and
Zhabotinsky, 2001). In this study, we concentrate on the
maintenance of the total amount of aCaMKII and the fraction
of phosphorylated aCaMKII. We assume that these variables
are directly correlated with synaptic efficacy, as shown in
previous theoretical studies (Lisman, 1989; Castellani et al,
2005).
Experimental results show that protein synthesis inhibitors
that are applied during the induction of L-LTPcan block L-LTP,
but if they are applied during the maintenance of L-LTP, they
do not reverse the potentiation (Frey et al, 1988; Fonseca et al,
2006). Similarly, several results show that the inhibition of
aCaMKII activity blocks the induction of L-LTP, but not its
maintenance (Malinow et al, 1989; Otmakhov et al, 1997).
These results might indicate that a basic assumption of the
model, that protein synthesis of new aCaMKII during
maintenance is required for maintenance, does not hold,
potentially invalidating the model. However, by simulating a
large, but not complete, inhibition of protein synthesis and
aCaMKII activity, we show a very different effect during the
induction and maintenance phases of L-LTP, which is not only
consistent with experimental observations but might also
account for an apparent discrepancy between different
experiments (Malinow et al, 1989; Otmakhov et al, 1997;
Sanhueza et al, 2007).
Results
A molecular model of aCaMKII interaction loop
The model that we propose for a translation-factor kinase loop
(Figure 1) is composed of two molecular components: (1) the
aCaMKII protein, which can be in one of three states: inactive,
active, or phosphorylated; (2) the CPEB1 translation factor,
which can be either in the phosphorylated or unphosphory-
lated state, binds to the CPE domain of the aCaMKII mRNA,
and represses its translation if unphosphorylated. These two
components interact through a closed loop. Here, we assume
that CPEB1 is phosphorylated by active and phosphorylated
aCaMKII (Atkins et al, 2004, 2005). Once CPEB1 is
phosphorylated by a kinase, it initiates the translation of a
new aCaMKII protein from aCaMKII mRNA through a
CPE-dependent polyadenylation process. In addition to new
protein synthesis, the proposed molecularmodel also assumes
that the aCaMKII protein undergoes degradation. This
degradation is a combination of several processes, including
Translational switch and synaptic plasticity
N Aslam et al
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protein turnover and permanent diffusion of non-bound
proteins out of synapses. All of the processes modeled herein
are complex, but we have approximated them by simpler
processes that maintain their key qualitative features. An
implicit assumption of this model is that aCaMKII levels are
related to synaptic efficacy, although this relationship might
not be linear.
Our model of aCaMKII activation and autophosphorylation
(Figure 1) assumes that the aCaMKII protein can reside in
three distinct states: an inactive state that cannot phosphor-
ylate substrates, an active state, which is obtained when
inactive aCaMKII binds to calcium–calmodulin ((Ca2þ)4–
CaM);thisactivestatecanphosphorylatesubstrates,including
other aCaMKII molecules. The third state is the phosphory-
lated state of aCaMKII, which is constitutively active,
independent of the level of (Ca2þ)4–CaM. The active
phosphorylated state can be dephosphorylated by a phospha-
tase. A faithful activation, autophosphorylation model of
aCaMKII is more complex than the three-state model assumed
here (Zhabotinsky, 2000; Lisman and Zhabotinsky, 2001); in
particular, it must take into account the structure of aCaMKII
holoenzymes. In our simulations, we assumed a non-
negligible basal level of inactive aCaMKII (10mM) (Chen
et al, 2005), whereas the basal concentrations for active
aCaMKII is considered to be negligibly small. The biochemical
reaction rates for the aCaMKII molecular loop are taken from
previous work (Kubota and Bower, 2001). For determining the
degradation rates of aCaMKII, we used constants consistent
with experimentally measuredrates(Ehlers, 2003). It has been
suggested that the aCaMKII loop is itself a bistable molecular
loop (Zhabotinsky, 2000; Lisman and Zhabotinsky, 2001)
However, the isolated aCaMKII molecular loop of our model
has only one stable steady state (Supplementary Figure S1) in
accordance with experimental results (Bradshaw et al, 2003).
Therefore, the bistability of this translation–phosphorylation
model originates from the overall structure of the aCaMKII–
CPEB1 molecular loop and not from the isolated aCaMKII
structure, in contrast to previous models (Zhabotinsky, 2000;
Lisman and Zhabotinsky, 2001).
The synthesis of new proteins in our proposed molecular
model is controlled by the phosphorylation of CPEB1
molecules. Once CPEB1 is phosphorylated, it initiates the
polyadenylation process,which leads to the synthesis of a new
aCaMKII molecule. In our model, the rate of new protein
synthesis is controlled by the fraction of phosphorylated
CPEB1 through a simple model of translation (see Materials
and methods). In our translation model (Figure 1), the
phosphorylatedCPEB1(YP)bindstothetranslationmachinery
(T) with a forward constant, kSYN1, a backward constant,
k(?12),andproducesanewproteinwithasynthesisrate,kSYN2.
Although this model is clearly a very simplified representation
of the translation processes, we presume that it captures the
qualitative features essential for this work. Although our
simplified model might be sufficient for capturing the
qualitative behavior of this loop, its simplified nature implies
that it may not account quantitatively forexperimental results.
We assume that aCaMKII is synthesized in its inactive state.
This model differs qualitatively from most previous models of
molecular bistability (Kholodenko, 2000; Tyson et al, 2003) in
that it does not assume conserved quantities of molecules and
includes both protein synthesis and degradation.
In this study, although we are primarily focused on the
maintenance phase of L-LTP, we modeled the induction of
L-LTP by using a brief pulse of increased (Ca2þ)4–CaM
concentration. This is used to mimic the high-frequency
stimulation (HFS) induction protocol of L-LTP. This is a very
CaMKII
(X)
CaMKII active
(XA)
Translation
(T)
CPEB
(Y)
CPEB
phosphorylated
(YP)
Degradation (rate ?1)
Degradation (rate ?2)
Degradation (rate ?3)
Protein synthesis (rate ksyn2)
Protein synthesis (rate ksyn1)
(Ca2+)4–CaM
CaMKII active,
phosphorylated (XA)
P
Figure 1
active and phosphorylated state, whereas CPEB1 can be in unphosphorylated or phosphorylated state. The aCaMKII molecule in each of the three states has a finite
turnover time. A new aCaMKII molecule is produced by CPEB1-dependent polyadenylation of aCaMKII mRNA, which is represented by translation stage (T).
The proposed model of the local aCaMKII synthesis through the aCaMKII–CPEB1 molecular loop. Here, the aCaMKII molecule can be in inactive, active or
Translational switch and synaptic plasticity
N Aslam et al
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simple approach that ignores many of the details of induction
protocols and the complex signal-transduction pathways that
are involved. However, as maintenance and not induction is
the focus of our work, we chose a simple model so as not to
obscure the essence of our results with details. Nevertheless,
the cost of this simple approach is that our results have
qualitative, but not quantitative, significance.
Bistable molecular switch
We next determined whether the kinase translation-factor loop
can operate as a bistable switch. We implemented the induction
of L-LTP through a (Ca2þ)4–CaM pulse as mentioned in a
previoussection.Intheabsenceofapulse(Figure2,solidlines),
the system stays in the lower steady state, wherein the total
concentration of aCaMKII is 10mM (Figure 2A,solid line),as the
restofthecellandthefractionofphosphorylatedaCaMKIIis0%
(Figure 2B, solid line).Onapplication of a pulse (10s),there is a
sustained increase in the total amount of aCaMKII to approxi-
mately twice the basal-level concentration (Figure 2A, dotted
line) and the fraction of phosphorylated aCaMKII is elevated
(Figure 2B, dotted line), but is far from saturation. These results
are consistent with experimental observations (Ouyang et al,
1997,1999).ThetemporaldynamicsdepictedinFigure2 clearly
show two phases of L-LTP: an early phase in which the
concentration of aCaMKII rapidly increases (30–40min),
followed by a persistent phase in which the concentration of
aCaMKII is maintained over a long period of time. The system
depicted here is bistable because we can provide stimulus of
different amplitudes, but they will converge to one of only two
possible stable states, depending on the amplitude and duration
of stimulus (Figure 2). We call these two stable states the ‘up’
and ‘down’ states. The sustained increase in the aCaMKII
amount and activity is believed to be linked with synaptic
potentiation.Previousmodelshaveinvestigatedtheconnections
between the signal transduction networks and changes in
synaptic efficacy during the induction phase (Castellani et al,
2001, 2005; Shouval et al, 2002; D’Alcantara et al, 2003), and
although similar components can be used here, it is beyond the
scope of this study.
Two key processes that might control the stability of the
proposed molecular model are protein synthesis and protein
degradation (Figure 1). In our simple model of degradation,
proteinturnoverischaracterizedbyturnoverrates.Tosimplify
further, we assume that the turnover rates for the different
states of aCaMKII are identical (l1¼l2¼l3), and to facilitate a
comparison with experimental results, we use the protein
degradation time constant, t¼1/l. In Figure 3, we present the
results of simulations with different degradation time con-
stants. At a large degradation time constant, the system is
monostable (Figure 3A). As degradation time constant
decreases, the system becomes bistable (Figures 3B and C)
and the difference between the ‘up’ and ‘down’ states
decreases (Figure 3A–C). Once the degradation rate is high
enough, the system loses its bistability and becomes mono-
stable (Figure 3D).
Bistability critically depends also on the rate of protein
synthesis. Protein synthesis is characterized in our simple
010002000
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Figure 2
through the application of HFS. The solid line represents the aCaMKII concentration at the basal level (without the application of pulse) and the dashed line represents
the aCaMKII concentration after the application of pulse. (A) Total aCaMKII concentration (mM) with a lower steady state at 10mM and an upper steady state at
B22mM.(B)FractionofactiveandphosphorylatedaCaMKIIwithalowersteadystateatB0andanuppersteadystateatB25%.Thisfigurealsoshowsthebistability
characteristics ofthe aCaMKII–CPEB loop atdifferent amplitudes ofthe (Ca2þ)4–CaMpulsefor 10sduration. Theseresultsshow thatevenwithdifferent amplitudes of
pulse stimulus, the aCaMKII–CPEB loop converges to the same up- or downregulated state, thus indicating that this system is bistable, that is, with a sufficiently strong
stimulus, it is upregulated and without enough stimulus it is downregulated.
Bistability characteristics of the aCaMKII–CPEB1-positive feedback loop. A brief (Ca2þ)4–CaM concentration pulse is used to mimic the induction of L-LTP
Translational switch and synaptic plasticity
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model byseveral parameters. In Figure 4, we show how one of
these parameters, kSYN1, affects the dynamics of our model.
These results are obtained at the aCaMKII turnover rate of 1h.
Here, we show that at low kSYN1, the molecular switch has a
monostable character (Figure 4A), which implies that at a
lowersynthesis rate, the new proteinsynthesis is not sufficient
to provide a replacement for protein loss due to degradation,
and therefore, the upregulated state of aCaMKII cannot be
maintained for a long period of time, leading to a gradual loss
of synaptic strength. However, at high kSYN1, the synthesis of a
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? = 111 h
? = 2.78 h
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A
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Figure3
of(Ca2þ)4–CaMpulseandthedashed linerepresentsaCaMKIIconcentrationonapplicationofabrief (Ca2þ)4–CaMpulse.Allsimulationsare carriedoutwithidentical
parameters, except the aCaMKII turnover rate (A) t¼111.0h (B) t¼2.78h (C) t¼0.4h (D) t¼0.27h. For a slow turnover rate, the switch is monostable (A), as the
turnover rate is increased, the switch becomes bistable (B and C), and at a faster turnover rate, the switch becomes monostable again (D).
BistableandmonostablecharacteristicsoftheaCaMKII–CPEB1-positivefeedbackloop.SolidlinerepresentsaCaMKIIconcentration,withouttheapplication
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Figure4
ThesolidlinerepresentsaCaMKIIatthebasalconditionandthedashedlinerepresentstheapplicationofpulse.Allsimulationsarecarriedoutwithidenticalparameters,
except the synthesis rate of new proteins (A) kSYN1¼0.0016mM?1sec?1(B) kSYN1¼0.0162mM?1sec?1(C) kSYN1¼0.056mM?1sec?1(D) kSYN1¼5.6mM?1sec?1.
The switch is monostable for slow protein synthesis rate (A), as the protein-synthesis rate is increased, the switch becomes bistable (B, C), and as the protein-synthesis
rate is further increased, it becomes monostable again (D).
BistableandmonostablecharacteristicsoftheaCaMKII–CPEB1-positivefeedbackloop.Thesimulationsareimplementedusingabrief(Ca2þ)4–CaMpulse.
Translational switch and synaptic plasticity
N Aslam et al
& 2009 EMBO and Macmillan Publishers LimitedMolecular Systems Biology 2009 5