of the Transcriptional Coactivator
CRTC1 from Synapse to Nucleus
Toh Hean Ch’ng,1Besim Uzgil,2Peter Lin,3Nuraly K. Avliyakulov,1Thomas J. O’Dell,4and Kelsey C. Martin1,5,6,*
1Department of Biological Chemistry
2Interdepartmental Program in Neuroscience
3Department of Microbiology, Immunology, and Molecular Genetics
4Department of Physiology
5Department of Psychiatry and Biobehavioral Sciences
6Integrated Center for Learning and Memory, David Geffen School of Medicine
University of California, Los Angeles, Los Angeles, CA 90095-1737, USA
Long-lasting changes in synaptic efficacy, such as
those underlying long-term memory, require tran-
scription. Activity-dependent transport of synapti-
cally localized transcriptional regulators provides a
direct means of coupling synaptic stimulation with
changes in transcription. The CREB-regulated tran-
scriptional coactivator (CRTC1), which is required
for long-term hippocampal plasticity, binds CREB
to potently promote transcription. We show that
CRTC1 localizes to synapses in silenced hippo-
campal neurons but translocates to the nucleus in
response to localized synaptic stimulation. Regu-
lated nuclear translocation occurs only in excitatory
neurons and requires calcium influx and calcineurin
activation. CRTC1 is controlled in a dual fashion
with activity regulating CRTC1 nuclear translocation
and cAMP modulating its persistence in the nucleus.
Neuronal activity triggers a complex change in
CRTC1 phosphorylation, suggesting that CRTC1
may link specific types of stimuli to specific changes
in gene expression. Together, our results indicate
that synapse-to-nuclear transport of CRTC1 dynam-
ically informs the nucleus about synaptic activity.
Hebbian and homeostatic forms of synaptic plasticity require
new gene expression for their persistence (Kandel, 2001; Turri-
giano, 2008). For stimulus-induced alterations in transcription
to occur, signals must be relayed from synapses to the nucleus
(Ch’ngand Martin, 2011; Cohen and Greenberg, 2008). Although
electrochemical processes permit extremely rapid signaling
between subcellular compartments in neurons, soluble signals
can also be transported from synapse to nucleus to trigger
changes in transcription (Ch’ng and Martin, 2011; Jordan and
Kreutz,2009; Thompson etal.,2004).Inducible transport oftran-
scriptional regulators from synapse to nucleus is a particularly
direct way of informing the nucleus about synaptic activity.
The transcription factor CREB plays a central role in many
forms of neuronal plasticity (Benito and Barco, 2010; Lonze
and Ginty, 2002). Stimuli that induce long-term plasticity activate
CREB-mediated transcription by triggering phosphorylation of
CREB at serine 133, leading to recruitment of CREB Binding
Protein (CBP) and transcriptional activation (Shaywitz and
Greenberg, 1999). Montminy and colleagues (Conkright et al.,
2003) and Labow and colleagues (Iourgenko et al., 2003) identi-
fied an additional regulator of CREB-mediated transcription in
pancreatic b islet cells, the CREB-regulated transcriptional
coactivator, CRTC (also known as transducer of regulated
CREB activity, TORC), whose activity is regulated by nucleocy-
toplasmic transport. In unstimulated cells, CRTC is phosphory-
lated (by salt-inducible kinase, SIK) and binds to 14-3-3 proteins
in the cytoplasm. Calcineurin-dependent dephosphorylation of
CRTC triggers its dissociation from 14-3-3 and subsequent
translocation into the nucleus. In the nucleus, CRTC binds the
bZIP domain of CREB (and other bZIP transcription factors)
and, in a manner that is independent of CREB phosphoryla-
tion, potently drives downstream gene expression by recruiting
TAFII130 and basal transcriptional machinery (Ravnskjaer
et al., 2007; Screaton et al., 2004). CRTC nuclear translocation
has been found to require coincident calcium and cAMP sig-
naling (Screaton et al., 2004).
Expression of dominant-negative forms of CRTC1 in CA1
neurons was reported to block the transcription-dependent
late phase of long-term potentiation (LTP), but not the early,
transcription-independent phase (Kova ´cs et al., 2007; Zhou
et al., 2006). Conversely, overexpression of CRTC1 in CA1 neu-
rons was found to lower the threshold for induction of late-phase
role for CRTC1 during the transcription-dependent phase of
Cell 150, 207–221, July 6, 2012 ª2012 Elsevier Inc. 207
Figure 1. Localization of CRTC1 to Dendrites and Synapses by Activity-Dependent Tethering to 14-3-3 ε
(A) Rat hippocampal neuron cultures (DIV 14–21) were immunostained with antibodies against panCRTC (green) and MAP2 (red).
(B) As in (A), but immunostaining with antibodies specific for CRTC1 (green), synaptotagmin (blue), and PSD95 (red) is shown.
(C) Mouse brains (5 weeks) were fractionated into synaptosomes (SYN) and PSDs and immunoblotted for CRTC1, synaptophysin, and PSD95.
(D) Cultured hippocampal cultures were treated with TTX (1 mM) or bicuculline (BIC; 40 mM) for 1 hr, fixed and stained with CRTC1 and PSD95 antibodies.
208 Cell 150, 207–221, July 6, 2012 ª2012 Elsevier Inc.
We followed up on these studies by determining the mecha-
nisms whereby specific types of synaptic stimulation trigger
CRTC1 nuclear import from distal synapses to the nucleus and
by characterizing the function of this nuclear translocation. We
show that CRTC1 localizes to dendrites and spines in electrically
silenced rodent hippocampal neurons, and translocates to the
nucleus in a calcium- and calcineurin-dependent manner
following glutamatergic synaptic transmission. CRTC1 is specif-
dent nuclear translocation occurs only in excitatory neurons.
Synaptic stimulation triggers complex changes in CRTC1 phos-
phorylation, suggesting that the phosphorylation state of CRTC1
may integrate specific types of activity to trigger specific
programs of CRE-dependent gene expression. We show that
in neurons, elevations in intracellular cAMP are not required for
CRTC1 nuclear import but rather regulate the persistence of
CRTC1 nuclear accumulation. siRNA knockdown of CRTC1
reveals that CRTC1 is required for the stimulus-induced regula-
tion of specific CREB target genes in a manner that is indepen-
dent of CREB (S133) phosphorylation. Together, our data
demonstrate that synaptically driven calcium influx triggers
nuclear translocation of CRTC1, whereas elevations in cAMP
regulate the persistence of nuclear CRTC1. In this way, CRTC1
dynamically informs the nucleus about synaptic activity to
mediate transcription-dependent forms of plasticity.
CRTC1 Localizes to Spines and Dendrites
et al., 2008; Watts et al., 2011). Using antibodies that specifically
recognize CRTC (Figure 1A; see Figure S1C available online) and
CRTC1 (Figures 1B–1D, S1A, and S1B) to stain cultured rat
hippocampal neurons (21 DIV), we detected immunoreactivity
in the soma, dendrites, and spines (Figures 1A and S1C). Triple
labeling with MAP2 (dendrites), synaptotagmin (presynaptic),
and PSD95 (excitatory postsynaptic) antibodies revealed locali-
zation throughout dendrites and at synapses (Figure 1B). Coloc-
alization analysis revealed that ?99% of PSD95-positive puncta
contained CRTC1; this association was further confirmed by
a positive Pearson’s correlation coefficient (r) (Figure S1D). To
further examine the synaptic localization of CRTC1, we fraction-
ated adult mouse brain into synaptoneurosome and postsyn-
aptic density (PSD) fractions and found that CRTC1 was present
in both (Figure 1C).
To determine whether the dendritic and synaptic localization
of CRTC1 was regulated by synaptic activity, we incubated
cultures (21 DIV) with the sodium channel blocker tetrodotoxin
(TTX, 1 mM, 4 hr), which blocks action potentials and thereby
silences neuronal cultures, or with the GABAAreceptor antago-
nist bicuculline (40 mM, 1 hr), which by blocking inhibition, drives
excitatory synaptic transmission in cultures. As shown in Fig-
ure 1D, whereas TTX did not significantly affect the spine local-
ization of CRTC1, incubation with bicuculline significantly
reducedCRTC1immunoreactivity inspines.Double-label immu-
nocytochemistry with antibodies against PSD95 and CRTC1 re-
vealed a 75% increase in the number of PSD95-positive puncta
that lack CRTC1 following bicuculline stimulation (Figure 1E).
Because phosphorylated CRTC1 is tethered in the cytoplasm
through interactions with 14-3-3 proteins in pancreatic b islet
cells (Screaton et al., 2004), we asked whether dendritic
CRTC1 colocalized with a particular 14-3-3 isoform in neurons.
Immunocytochemistry with antibodies recognizing 14-3-3 b, g,
ε, s, h, t, and z isoforms revealed that the ε isoform was present
in dendritic spines (Figure S1E). Double-label experiments re-
vealed striking colocalization between CRTC1 and 14-3-3 ε in
dendrites and spines (Figure 1F). Moreover, pull-down ex-
periments with GST-14-3-3 ε revealed an activity-regulated
interaction with CRTC1: binding was detected in electrically
silenced neurons but dramatically reduced following bicuculline
stimulation. Mutation at the binding pocket of 14-3-3 ε (K49E)
completely abolished its interaction with CRTC1 (Figure 1G).
These findings suggest that CRTC1 undergoes activity-
regulated tethering at synapses by binding to 14-3-3 ε.
CRTC1 Undergoes Activity-Dependent Nuclear
Accumulation in Excitatory Neurons
Confocal imaging of the cell body in basal, TTX, and bicuculline-
stimulated cultured neurons revealed that CRTC1 was excluded
from the nucleus under basal and TTX conditions but accumu-
lated in the nucleus following incubation with bicuculline (Fig-
ure 2A). Blocking excitatory synaptic transmission with the
AMPA receptor antagonist NBQX completely blocked CRTC1
nuclear translocation, whereas preincubation with the NMDA
receptor antagonist APV significantly inhibited nuclear accumu-
lation, indicating that activation of both AMPA and NMDA re-
ceptors contributes to CRTC1 synapse-to-nuclear transport
(Figure S2B). Removal of calcium from the extracellular media
or inhibition of L-type voltage-gated calcium channels (LVGCCs)
with nimodipine completely blocked nuclear accumulation of
CRTC1, consistent with a requirement for influx of extracellular
calcium through LVGCCs (Figure 2A). The ability of bicuculline
to drive nuclear accumulation correlated with the synaptic
connectivity of the neurons; accumulation was observed after
14 DIV, but not at 7 DIV, when neuronal cultures have fewer
synaptic connections (Figure S2A).
To monitor the persistence of stimulus-induced CRTC1
nuclear translocation, we incubated neurons with bicuculline
(E) The percentage of PSD95-positive synapses lacking CRTC1 immunoreactivity (**p < 0.01 relative to basal control) is presented.
(F) Hippocampal neurons were immunostained with antibodies against CRTC1 (green) and 14-3-3 ε (red).
(G) GST-14-3-3 ε (WT) and binding mutant (K49E) were incubated with protein lysates from hippocampal cultures pretreated with TTX (1 mM) and CsA (5 mM) for
2hr or with bicuculline (40mM)and forskolin (25 mM)for 15min. GST pull-downs were immunoblotted with CRTC1, GST, TUJ1, and 14-3-3 ε antibodies,and blots
were stained with Sypro Ruby to verify protein concentration and purity.
Scale bars, 10 mm. For related data see also Figure S1.
Cell 150, 207–221, July 6, 2012 ª2012 Elsevier Inc. 209
Figure 2. Activity-Dependent Nuclear Translocation of CRTC1 in Excitatory Hippocampal Neurons
(A)Bicuculline(BIC;40mM,1hr)wasaddedtountreated(basal) hippocampal culturesortohippocampal culturespretreatedwithAPV(100mM),nimodipine (NIM;
10 mM), or to cultures in a calcium-free Tyrode’s solution. After staining with CRTC1 (green) and MAP2 (red) antibodies and with Hoechst nuclear dye (blue,
merged), the nuclear-to-cytoplasmic ratio of CRTC1 was quantified (**p < 0.001 relative to basal).
(B) Hippocampal cultures were incubated with bicuculline (40 mM) for 10 min before recovery (0.5–24 hr) in the continued presence or absence of bicuculline.
Neurons were fixed and immunostained with CRTC1 and MAP2 antibodies and with Hoechst nuclear dye. The nuclear-to-cytoplasmic ratio of CRTC1 was
quantified (**p < 0.001 compared to TTX).
210 Cell 150, 207–221, July 6, 2012 ª2012 Elsevier Inc.
for 24 hr either with or without bicuculline. Twenty minutes after
bicuculline removal, the concentration of CRTC1 in the nucleus
returned to basal levels (Figure 2B). CRTC1 remained in the
nucleus as long as bicuculline was present, even after 24 hr of
continuous stimulation (Figure 2B). These results indicate that
We observed that in 15%–20% of our hippocampal cultures,
CRTC1 did not translocate to the nucleus following bicuculline
stimulation. As shown in Figure 2C, double-label experiments
with GAD67 to label inhibitory neurons and CamKIIa to label
excitatory neurons revealed that although CRTC1 was present
in both cell types, bicuculline-induced nuclear translocation
occurred exclusively in excitatory neurons.
We next asked whether nuclear CRTC1 resulted from nucleo-
cytoplasmic transport or from new synthesis of CRTC1. Incuba-
tion of neurons with the protein synthesis inhibitor emetine prior
to and during bicuculline stimulation did not prevent CRTC1
nuclear translocation (Figure S2C), and no change in the total
concentration of CRTC1 was observed following TTX or bicucul-
line stimulation (Figure S2D). Thus, bicuculline induces nuclear
import of pre-existing CRTC1.
CRTC1 Translocates to the Nucleus of Hippocampal
Neurons following Induction of LTP in Organotypic Slice
Following up on earlier studies showing nuclear accumulation of
CRTC1 in acute hippocampal slices after induction of L-LTP
(Zhou et al., 2006), we asked whether we could detect loss of
CRTC1 from dendrites and synapses and accumulation in the
nucleus following LTP induction in organotypic hippocampal
slice cultures (16–18 DIV). To induce chemical LTP (cLTP), we
incubated slice cultures for 60 min in Mg2+-free artificial cerebro-
spinal fluid (ACSF) supplemented with rolipram, forskolin, and
picrotoxin (Kopec et al., 2006). As shown in Figure 3A, in unsti-
mulated slice cultures CRTC1 was present in stratum radiatum
dendrites but was excluded from the nucleus. Following
cLTP stimulation, robust nuclear CRTC1 immunoreactivity was
detected in all three cell body layers (dentate, CA3, and CA1).
Nuclear accumulation was accompanied by a loss of CRTC1
immunoreactivity in MAP2-positive dendrites in the stratum
radiatum (Figure 3A, white arrowheads). To complement the
studies in organotypic slice cultures, we briefly depolarized neu-
rons with KCl in acute hippocampal slices, which not only
triggered nuclear translocation but also resulted in a loss of
immunoreactivity in the stratum radiatum (Figure S3A).
CRTC1 Nuclear Translocation in Acute Hippocampal
Slices Requires Synaptic Activity
Zhou et al. (2006) showed that CRTC1 underwent translocation
into CA1 pyramidal nuclei in acute hippocampal slices following
4 3 100 Hz tetanic stimulation, which induces transcription-
dependent L-LTP, but not following a single 100 Hz tetanic stim-
ulus, which induces transcription-independent E-LTP. However,
these experiments were performed in the presence of bicucul-
line, which we found was sufficient on its own to drive CRTC1
nuclear import in hippocampal slices (data not shown). To
more specifically test the requirement for synaptic activity to
drive CRTC1 nuclear translocation in acute hippocampal slices,
we stimulated Schaffer collateral fiber synapses onto CA1
pyramidal cells using multiple trains of theta frequency (5 Hz)
stimulation. As shown in Figure S3B, this stimulation paradigm
triggered nuclear translocation in CA1 neurons, but not in CA3
neurons. Because Schaffer collateral fiber stimulation not only
activates synapses onto CA1 pyramidal cells but also triggers
antidromic action potentials in CA3 pyramidal cells, this finding
suggested that synaptic activation is specifically required for
CRTC1 nuclear translocation. To more rigorously test this possi-
bility, we delivered the same pattern of theta frequency stimula-
tion to the alveus to selectively trigger antidromic action poten-
tials in CA1 pyramidal cells in slices in which excitatory
synaptic transmission was blocked with the broad-spectrum
ionotropic glutamate receptor antagonist kynurenate (3 mM).
As shown in Figure 3B, postsynaptic action potentials in the
absence of excitatory synaptic transmission failed to induce
nuclear translocation of CRTC1. These results indicate that syn-
neuronal depolarization is not sufficient.
CRTC1 Translocates Specifically from Stimulated
Synapses to the Nucleus
To specifically monitor the transport of CRTC1 from stimulated
subsets of synapses to nucleus, we performed two sets of ex-
periments. In the first, we overexpressed CRTC1 fused to the
photoconvertible fluorescent protein dendra2 in cultured hippo-
campal neurons (Figure 4A). In unstimulated neurons, only low
levels of CRTC1-dendra2 were detected in the nucleus. A brief
UV illumination of distal dendrites (?100–200 mm from soma)
converted the dendra2 signal from green to red. Using time-
lapse imaging, we followed the accumulation of both the native
green and photoconverted signals in the cell body over a period
of 30 min postconversion. Our results revealed that the photo-
converted (red) dendritic CRTC1 underwent stimulus-induced
translocation into the nucleus (Figures 4Ai–4Aiii). We also
observed that the rate of nuclear accumulation of CRTC1-
dendra2 is fastest during the first 10 min after stimulation,
consistent with stimulus-induced active retrograde transport
We next asked whether endogenous CRTC1 underwent
synapse-to-nucleus translocation following local stimulation.
To do this, we cultured neurons on gridded coverslips and
transduced the neurons with a lentivirus expressing eGFP to
visualize the entire dendritic arbor of individual neurons. We
locally UV uncaged MNI glutamate (or vehicle, in controls) at
(C) Hippocampal cultures were incubated with bicuculline (40 mM) for 1 hr, fixed, and double labeled with CRTC1 (green) and GAD67 (red) or CRTC1 (green) and
CamKIIa (red) antibodies.White arrows indicate presynaptic GAD67-positive puncta incontact withthe somaof an excitatoryneuron.Thetotalconcentrationsof
somatic CRTC1, and the nuclear-to-cytoplasmic ratio of CRTC1, were quantified in excitatory and inhibitory neurons (**p < 0.001 relative to excitatory neurons;
n.s., not significant).
Scale bars, 10 mm. See also Figure S2.
Cell 150, 207–221, July 6, 2012 ª2012 Elsevier Inc. 211
distal dendrites of GFP-expressing neurons, and 10–30 min later
fixed and immunolabeled with anti-CRTC1 and MAP2 anti-
bodies. As shown in Figure S4, a brief UV pulse at a distal site
uncaged sufficient glutamate to trigger a robust dendritic
calcium signal and, as shown in Figure 4Bi, also significantly
increased the concentration of CRTC1 in the nucleus as com-
pared to controls. These data indicate that stimulation of distal
synapses is sufficient to trigger nuclear accumulation of CRTC1.
Examination of CRTC1 immunoreactivity in dendritic seg-
ments of neurons following uncaging revealed that glutamate
uncaging resulted in a loss of CRTC1 in local dendritic segments
compared to dendrites receiving mock uncaging. Furthermore,
loss of dendritic CRTC1 immunoreactivity was branch specific;
local uncaging at one branch triggered a loss of CRTC1 from
that branch without changing CRTC1 immunoreactivity in other
dendritic branches from the same neuron (Figure 4Bii). The
finding that CRTC1 loss was specific to the site of stimulation
even though uncaging produced a much broader depolarization
(Figure S4) also demonstrates a requirement for synaptic stimu-
lation, as opposed to depolarization, in CRTC1 nuclear import.
Figure 3. Synapse-to-Nucleus Transloca-
tion of CRTC1 in Organotypic Slice Cultures
and Acute Hippocampal Slice Preparations
(A) cLTP was induced in organotypic hippocampal
slice cultures, which were then fixed and im-
munolabeled with antibodies for CRTC1 (green)
and Hoechst nuclear dye (blue). Representative
confocal sections at 103 magnification (scale bar,
10 mm) and 403 magnification (scale bar, 100 mm)
of the CA1 cell body layer are shown. Arrowheads
indicate presence of CRTC1 in dendrites. Dashed
box indicates CA1 cell body layer shown in high
magnification in right panels.
(B) Theta pulse stimulation (TPS; five trains of 5 Hz
stimulation; 30 s duration with 30 s intertrain
interval) was delivered to Schaffer collateral
fibers in the stratum radiatum (R, orthrodromic
stimulation) or directly to the alveus to stimulate
the axons of the CA1 pyramidal neurons (anti-
dromic stimulation). Traces show examples of
evoked antidromic and postsynaptic responses.
After stimulation, slices were collected and im-
CA1 region of acute hippocampal slices. Scale
bar, 30 mm. Panels show an unstimulated control
slice (left) and slices where TPS was delivered to
the alveus (middle) or to the Schaffer collateral
fibers in stratum radiatum (right). P, stratum
See also Figure S3.
Calcineurin Is Required for CRTC1
dephosphorylation by calcineurin has
been shown to trigger its release from
14-3-3 and subsequent CRTC2 translo-
cation into the nucleus (Conkright et al.,
synapse-to-nuclear translocation in neurons. As shown in Fig-
ure 5A, preincubation of cultured hippocampal neurons with
the calcineurin antagonist cyclosporin A (CsA; 30 min; 5 mM)
completely blocked nuclear translocation of CRTC1 induced
active calcineurin (HA-CnA*) was sufficient to drive nuclear
import of CRTC1 even when the neurons were silenced with
TTX. In contrast, overexpression of full-length calcineurin (HA-
FL-CnA) was unable to initiate nuclear import of CRTC1 in the
absence of neuronal activity (Figure 5B).
Elevations in Intracellular cAMP Increase the
Persistence of Nuclear CRTC1
Nuclear translocation of CRTC2 has been reported to require
coincident elevations in calcium and cAMP in nonneuronal cells
(Screaton et al., 2004), triggering coincident calcineurin activa-
tion and SIK inactivation. To study the role of cAMP during
CRTC1 translocation in neurons, we briefly incubated dissoci-
ated cultures with forskolin (25 mM, 10 min) to activate adenylyl
cyclase. Because forskolin also increases excitability of cultured
212 Cell 150, 207–221, July 6, 2012 ª2012 Elsevier Inc.
hippocampal neurons (Hoffman and Johnston, 1998), we per-
formed these experiments in the presence or absence of TTX.
As shown in Figure 5C, whereas forskolin induces nuclear accu-
mulation of CRTC1, it does so only in the presence of neuronal
activity; TTX-silenced neurons did not undergo forskolin-
induced CRTC1 nuclear translocation (Figure 5C). Our studies
requires calcineurin (Figure 5A), extracellular calcium (Fig-
ure S5A), and LVGCC (Figure S5B).
We next asked whether the cAMP-PKA pathway was required
for CRTC1 nuclear translocation during bicuculline-induced
synaptic activation by incubating neurons in pharmacological
agents that block adenylyl cyclase activity (SQ22536; 20 mM),
antagonize PKA (KT5720; 2 mM), or competitively inhibit cAMP
(Rp-cAMP; 0.5 mM). As shown in Figures 5D and S5C, none of
these agents blocked the nuclear accumulation of CRTC1 in
neurons induced by bicuculline, indicating that increases in
cAMP are not required for CRTC1 nuclear import.
cAMP blocks the rephosphorylation of CRTC1 by inhibiting
AMPK or SIK (Conkright et al., 2003; Mair et al., 2011; Screa-
ton et al., 2004). This suggested to us that whereas increases
in cAMP might not be required for the initial import of CRTC1
from synapse to nucleus, they might increase the persistence
of nuclear CRTC1. We tested this idea by stimulating neurons
briefly with bicuculline and forskolin for 10 min, followed by a
quick washout and incubation in TTX and forskolin for another
15–30 min (Figure 5E). As shown in Figure 5F, addition of
forskolin (in the presence of TTX) following the initial bicucul-
line stimulation prolongs the nuclear accumulation of CRTC1,
presumably by preventing the rephosphorylation CRTC1.
To examine the specific role of AMPK or SIK in this experi-
ment, we stimulated neurons with bicuculline, and then al-
lowed the neurons to recover in the presence of dorsomorphin
dihydrochloride (DM; 20 mM), an inhibitor of both AMPK and
SIK activity (Sasaki et al., 2011). DM prolonged CRTC1 pres-
ence in the nucleus (Figure S5D), consistent with AMPK or
SIK rephosphorylating CRTC1 and promoting rapid nuclear
CRTC1 Undergoes Differential Patterns of Regulated
Phosphorylation and Dephosphorylation
To gain further insight into the mechanisms whereby stimulation
triggers CRTC1 translocation from synapse to nucleus, we
performed immunoblots of cultured neurons silenced with TTX
(1 mM; 1 hr) or stimulated with either bicuculline (40 mM;
10 min) or forskolin (25 mM; 10 min). As shown in Figure 6A,these
experiments revealed large, activity-dependent shifts in the
molecular weight (MW) of the protein. When neuronal cultures
were silenced with TTX, CRTC1 was ?10–15 kDa larger in MW
than it was in neuronal cultures that were stimulated with either
bicuculline or forskolin.
The coding region of mouse CRTC1 contains 146 serine,
threonine, and tyrosine residues (approximately 1 in 4.3 resi-
dues). Based on this and previous work on CRTC2 by Screaton
et al. (2004), we reasoned that the MW shift might be due
to phosphorylation of CRTC1. Incubation of lysates with calf
intestinal phosphatase shifted CRTC1 to a much lower MW,
suggesting that the shifts in MW resulted primarily from regu-
lated phosphorylation and dephosphorylation (Figure S6B).
We further used a CRTC1 antibody that specifically recognizes
the phosphorylated serine residue at S151 (Figure S6A). This
antibody primarily detected only bands that were higher
in MW, which likely correspond to phosphorylated CRTC1
(Figures 6A and S6A). When lysates were incubated with the
calcineurin inhibitor CsA, neither bicuculline nor forskolin
induced a decrease in MW, or a dephosphorylation of serine
151, consistent with calcineurin-dependent dephosphoryla-
tion of CRTC1 in response to stimulation (Figure 6A). To com-
plement our studies in cell culture, we also analyzed the
hippocampal acute slice after theta pulse stimulation of the
Schaeffer collateral (stimulation as described in Figure 3B) via
western blots and observed a significant reduction in the
levels of CRTC1 that was phosphorylated at serine 151
We next examined the activity-dependent phosphorylation
As shown in Figure 6B, in TTX-silenced cultures, CRTC1 ran as
a series of discrete spots (green) clustered toward the acidic
pH 3 isoelectric point, and running at approximately 75 kDa.
As a reference, we costained the 2D gels with antibodies that
detect the neuron-specific class III b-tubulin (TUJ1; 55 kDa; pI
4.88, red). Ten minutes of stimulation with bicuculline triggered
a dramatic shift in CRTC1 immunoreactivity toward the more
basic, pH 11 isoelectric point, and a decrease in MW. Ten
minutes of stimulation with forskolin also triggered a shift toward
more basic and lower MW spots, although the extent of dephos-
phorylation was not as great as with bicuculline stimulation.
When lysates were incubated with l phosphatase, CRTC1
immunoreactivity converged on a cluster of spots closer to
pH 11 isoelectric point. The complete loss of phospho-MAP
kinase immunoreactivity following incubation with l phospha-
tase treatment demonstrates the efficacy of the dephosphoryla-
tion (Figure S6D). Together, these data indicate that CRTC1
undergoes a complex change in phosphorylation in response
to stimuli. The finding that phosphatase treatment of lysates
did not collapse CRTC1 to a single spot indicates that, whereas
dephosphorylation accounts for the majority of change in pI and
MW following stimulation, CRTC1 likely undergoes additional
posttranslational modifications (Liu et al., 2008; Jeong et al.,
Phosphorylation of S151 by SIK has been reported to be
necessary for 14-3-3 binding and cytoplasmic anchoring in
nonneuronal cells (Screaton et al., 2004). As described above,
we found that stimulation of hippocampal neurons with bicu-
culline or forskolin triggered calcineurin-dependent S151
dephosphorylation (Figure 6A). To test whether S151 dephos-
phorylation was sufficient to drive nuclear translocation, we
generated a HA-tagged CRTC1 mutant in which S151 was
changed to an alanine (CRTC1S151A) and, thus, could not be
phosphorylated. This mutant localizes constitutively to the
nucleus in mouse hypothalamic GT1-7 cells (Altarejos et al.,
2008). However, when expressed in primary cultured hippo-
campal neurons, the CRTC1S151Amutant was excluded from
the nucleus in basal or TTX-silenced neurons but underwent
bicuculline-induced translocation into the nucleus (Figure 6C).
These results indicate that elevations in intracellular calcium
Cell 150, 207–221, July 6, 2012 ª2012 Elsevier Inc. 213
Figure 4. Synapse-to-Nucleus Translocation of CRTC1 in Hippocampal Neurons
(Ai) Hippocampal neurons were transfected with CRTC1-dendra2 for 14–18 hr before incubation in Tyrode’s solution in the presence or absence of calcium. After
taking baseline images of CRTC1-dendra2 expression, cultures were incubated with stimuli (Leptomycin B, 10 nM; bicuculline 40 mM; forskolin 25 mM) or were
unstimulated (Leptomycin B; 10 nM), and specific dendritic branches expressing the dendra2 construct were photoconverted from green to red with a UV pulse
laser. Nuclearred dendra2signalwasimaged every5minfor 30min. (Aii)Thepercentincrease ofphotoconverted dendra2signal inthenucleus wasquantifiedas
comparedtobaselinevalues.(Aiii)Groupdata (**p<0.001 relativetonostimulation)areshown.(Aiv)Therateatwhichphotoconverted reddendra2 fusionprotein
entered the nucleus was quantified and plotted in 10 min intervals.
(Bi) Hippocampal neurons were transduced with lentivirus expressing GFP. Glutamate was uncaged at distal dendrites of GFP-expressing neurons, followed by
fixation and staining for MAP2 (green),CRTC1 (red), and Hoechst nuclear dye (blue). The red box indicates region of local uncaging. Neurons were identified after
214 Cell 150, 207–221, July 6, 2012 ª2012 Elsevier Inc.
and cAMP trigger dephosphorylation of S151 in CRTC1, but
consistent with the 2D gel analysis, which reveals that multiple
residues undergo regulated dephosphorylation, S151 dephos-
phorylation on its own is not sufficient for nuclear import in
CRTC1 Is Required for Induction of Specific CREB Gene
To address the function of activity-dependent CRTC1 nuclear
translocation, we reduced CRTC1 expression in hippocampal
neurons with CRTC1 siRNAs (Figure S7A). We then used an
established protocol to study induction of CREB-induced gene
expression in cultured hippocampal neurons, in which cultures
are silenced with TTX for 6 hr, and TTX is then withdrawn,
leading to robust action potential firing and induction of several
immediate-early genes (Saha et al., 2011). This protocol also
triggered nuclear translocation of CRTC1 (Figure S7B). As
shown in Figures 7A and S7C, in cultures in which CRTC1
concentrations were reduced by ?60%, the induction of five
CREB target genes, including cfos, arc, egr4, zif268, and
cyr61, was significantly reduced compared to cultures receiving
nontargeting siRNAs. In contrast the induction of other CREB
target genes, including nr4a3, BDNF, shank3, and dusp1, was
not affected by the reduction in CRTC1 concentrations at the
time point examined (30 min after TTX withdrawal). We also
asked whether silencing of CRTC1 had any effect on CREB
phosphorylation. As shown in Figure 7B, TTX withdrawal in-
duced equivalent CREB phosphorylation at serine133 in cultures
treated with CRTC1 or nontargeting siRNAs. That CRTC1
knockdown inhibited the induction of cfos, arc, egr4, zif268,
and cyr61 mRNAs without any effects on CREB phosphorylation
indicates that CRTC1 nuclear translocation, rather than serine
133 phosphorylation, is critical to the stimulus-induced expres-
sion of these CREB target genes.
The results of our studies indicate that the transcriptional regu-
lator CRTC1 undergoes activity-dependent trafficking from
dendrites and synapses to the nucleus in hippocampal neurons.
CRTC1 nuclear accumulation is tightly coupled to stimulation,
with synaptic activity rapidly triggering translocation of CRTC1
from synapse to nucleus and with CRTC1 remaining localized
in the nucleus as long as excitatory synaptic activity or cAMP
levels remain elevated. These data indicate that nuclear accu-
mulation of CRTC1 is a sensitive monitor of synaptic and neu-
romodulatory activitythatdynamically informs thenucleusabout
activity received at synapses. Because the nuclear translocation
does not require any transcription or translation, it is also a very
rapid marker of activity.
The Relationship between CREB and CRTC1 in
Establishing Long-Term Memory
Studies in multiple systems have uncovered a central role for
CREB-dependent transcription in the conversion of short-term
to long-term plasticity and memory (Silva et al., 1998; Kauffman
et al., 2010; but see also Balschun et al., 2003; Perazzona et al.,
2004). Previous studies have focused primarily on activation of
CREB by phosphorylation at serine 133 (pCREB133), and
pCREB133 immunoreactivity is often used as a proxy for
long-term plasticity and memory. Increasing evidence, how-
ever, indicates that CREB phosphorylation at serine 133 does
not always correspond to transcriptional activation, raising the
question of whether additional means of activating CRE-driven
transcription operate during plasticity and memory (Bito et al.,
1996; Impey et al., 1996; Kornhauser et al., 2002). Transcrip-
tional activation mediated by CRTC nuclear import, which
can dramatically increase CRE-driven gene expression in the
absence of serine 133 phosphorylation, provides one such
mechanism (Conkright et al., 2003; Iourgenko et al., 2003;
Screaton et al., 2004).
How the phosphorylation state of CREB relates to CRTC1-
induced transcriptional activation following stimulation in neu-
rons remains unclear. One possibility is that distinct states of
CREB phosphorylation, on serine 133 as well as other residues
(Kornhauser et al., 2002), coupled with CRTC1 activation, may
allow CREB to transcribe specific subsets of genes in response
to distinct stimuli. Our data showing that CRTC1 undergoes an
ylation at multiple residues (Figure 6) suggest a degree of
complexity that could contribute significantly to diverse tran-
scriptional responses. Thus, distinct stimuli may elicit distinct
patterns of CRTC1 phosphorylation to allow recruitment of
distinct bZIP transcription factors, thereby conferring selectivity
of CREB-mediated gene expression to generate distinct pro-
grams of gene activation. It will be of great interest to map
out the specific residues that undergo regulated changes in
phosphorylation, and to then determine how phosphorylation/
dephosphorylation of each site alters downstream gene
Synapse-to-Nuclear Trafficking of CRTC1 in Neurons
In addition to potentially contributing to the specificity of
CREB-dependent transcriptional responses, activity-dependent
synapse-to-nucleus translocation of CRTC1 may preserve
spatial information about the initial site of stimulation. Thus,
stimuli that lead to CREB phosphorylation do so by activating
second messenger cascades that spread throughout the cell.
In this mode of signaling, information about the spatial location
of the originating stimulus is lost. However, stimuli that promote
CRTC1 translocation from synapse to nucleus do so by
dendrites adjacent to the region of photouncaging of glutamate was quantified (region A). As a control, a randomly selected branch of dendrite adjacent to the
areaofactivationwasalsoselected (regionB),and theamountofCRTC1 wasquantified.Theratioofregion Atoregion Bwasdeterminedand plotted asascatter
plot (**p < 0.05, paired Student’s t test). A ratio of one indicates equal amounts of CRTC1 in the glutamate uncaged and adjacent control dendrite. Group data of
the percent increase of CRTC1 in control relative to the uncaged dendrite for mock and glutamate-uncaged neurons (**p < 0.05, paired Student’s t test) are
Scale bars, 10 mm. For related data, see also Figure S4.
Cell 150, 207–221, July 6, 2012 ª2012 Elsevier Inc. 215
Figure 5. Nuclear Translocation of CRTC1 Requires Activation of Calcineurin; cAMP Regulates the Persistence of CRTC1 in the Nucleus
(A) Hippocampal cultures were pretreated with CsA (5 mM) for 4 hr prior to a 1 hr stimulation with bicuculline (BIC; 40 mM) or with forskolin (FSK; 25 mM). Neurons
were fixed and immunostained with antibodies against MAP2 (red), CRTC1 (green), and Hoechst nuclear dye (blue, merged), and the mean nuclear-to-
cytoplasmic ratio was quantified (**p < 0.001 relative to nonstimulated but CsA-treated sample).
216 Cell 150, 207–221, July 6, 2012 ª2012 Elsevier Inc.
triggering loss of CRTC1 specifically from stimulated synapses.
Our experiments using local glutamate uncaging followed by
CRTC1 immunocytochemistry (Figure 4B) indicate that the loss
of CRTC1 is confined to the stimulated dendrite. Future experi-
ments aimed at resolving the loss of CRTC1 from individual
synapses may provide insight into the nature of the unit of stim-
ulation that is required for long-term changes in synaptic
cAMP Regulates CRTC1 Nuclear Persistence
Our experiments indicate that elevations in intracellular cAMP,
in the absence of neuronal activity, are not sufficient to trigger
CRTC1 nuclear translocation in neurons (Figure 5C). Moreover,
inhibiting cAMP function in cultured neurons did not block
bicuculline-induced nuclear translocation of CRTC1 (Figures
5D and S5C), strongly arguing that the cAMP-PKA pathway is
not necessary for the initial translocation of CRTC1 to nucleus.
Forskolin-induced CRTC1 nuclear translocation in dissociated
cultures likely results from cAMP-induced increases in neuronal
excitability (Hoffman and Johnston, 1998; Madison and Nicoll,
1986). Our findings stand in contrast to several other pub-
lished reports that indicate that cAMP can induce translocation
in a calcineurin-independent mechanism (Bittinger et al., 2004).
We demonstrate that in neurons cAMP regulates CRTC1
nuclear persistence, rather than CRTC1 nuclear import (Fig-
ure 5F). We propose that cAMP, by inactivating SIK and/or
AMP kinases (Katoh et al., 2004, 2006), prevents the rapid
rephosphorylation of CRTC1, which in turn prolongs CRTC1
This observation has important implications about the func-
tion of neuromodulators such as dopamine and norepinephrine,
both of which elevate intracellular cAMP, in long-term memory
formation. Our findings suggest that synaptic stimuli activate
calcineurin to trigger the nuclear translocation of synaptic
CRTC1. CRTC1 remains in the nucleus as long as synaptic
stimulation persists, but its nuclear persistence can be main-
tained in the absence of activity if cAMP levels are elevated.
Relevant to this hypothesis, norepinephrine and dopamine
concentrations are elevated in the hippocampus for up to 5 hr
following strong tetanic stimulation (Neugebauer et al., 2009).
From a learning perspective, this would imply that activation
of modulatory neurotransmission following a stimulus increases
the transcriptional changes induced by that stimulus. This idea
is supported by a wealth of literature showing that emotional
arousal, acting through neuromodulators like norepinephrine
and dopamine,enhances long-term
(McGaugh, 2006; Rossato et al., 2009; Navakkode et al.,
2007; O’Dell et al., 2010). Nuclear translocation of CRTC1 in
response to glutamatergic synaptic activity, followed by mainte-
nance of CRTC1 in the nucleus in response to neuromodulatory
neurotransmission, provides a molecular mechanism for these
The finding that siRNA knockdown of CRTC1 in cultured
hippocampal neurons inhibits the induction of specific CREB
targets in response to TTX withdrawal, including cfos, arc,
Egr4, zif268, and cyr61 (Figures 7A and S7C), indicates that
CRTC1 has a critical function in the transcriptional response to
neuronal activity. These changes are particularly remarkable
because the siRNA knockdown is incomplete (reduces levels
of CRTC1 to ?40%). Moreover, we found that CREB phosphor-
ylation at serine 133 following TTX withdrawal was not altered by
CRTC1 knockdown (Figure 7B), indicating that CREB phosphor-
ylation on its ownisinsufficient to drivefull expression of specific
CRE-containing genes, and that activity-dependent CRTC1
nuclear translocation is required.
Taken together, the results of our studies raise the possibility
that excitatory synaptic activity and neuromodulators contribute
to dynamic changes in gene expression in mechanistically
distinct ways, with synaptic glutamatergic stimulation triggering
nuclear import of CRTC1 and neuromodulators regulating its
duration in the nucleus. Given the complexity of the stimulus-
induced changes in CRTC1 phosphorylation, it is likely that
many other types of neuronal activity might differentially influ-
ence CRTC1-dependent gene expression and thereby trigger
distinct types of CREB-dependent memory over distinct time
Neuron Culture and Pharmacological Treatments
All experiments were performed using approaches approved by the UCLA
Institutional Animal Care and Use Committee. Rodent hippocampal neurons
were cultured for 2–4 weeks as described in Extended Experimental Proce-
dures. All pharmacological manipulations of neurons are also described in
Extended Experimental Procedures.
(B) Full-length (HA-FL-CnA) or constitutively activated calcineurin (HA-CnA*) fused to an HA epitope tag was transiently transfected into hippocampal cultures.
After 24 hr, transfected cultures were preincubated with either TTX (1 mM) for 1 hr or with bicuculline (40 mM) for 10 min, fixed, and immunostained with antibodies
against CRTC1 (green), HA (red), MAP2 (cyan), and the Hoechst nuclear dye (blue, merged). The nuclear-to-cytoplasmic ratio of CRTC1 was quantified
(**p < 0.001 when compared to HA-FL-CnA).
(C) Hippocampal neurons were stimulated for 10 min with forskolin (25 mM) in the presence or absence of TTX (1 mM, 1 hr pretreatment). The nuclear-to-
cytoplasmic ratio of CRTC1 was quantified (**p < 0.001 relative to basal level).
(D) Hippocampal neurons were pretreated with Rp-cAMP (0.5 mM) or KT5720 (2 mM) for 30 min prior to stimulation with bicuculline (40 mM, 10 min). The nuclear-
to-cytoplasmic ratio of CRTC1 was quantified (**p < 0.001 relative to no BIC-treated controls).
(E)Neuronswerestimulatedwitheither BIC+FSK orTTX for 10min,washed and incubated withmedia containing TTX alone,BIC +FSKor TTX +FSKfor another
15 or 30 min (pooled data). A flow chart and time course of the treatment are included for all four stimulation paradigms (i–iv).
(F) After fixation and immunostaining, the nuclear-to-cytoplasmic ratio of CRTC1 was quantified. For all experiments, neurons were immunostained with CRTC1
(green), MAP2 (red), and Hoechst nuclear dye (blue). The normalized nuclear-to-cytoplasmic ratio of CRTC1 relative to TTX-treated samples was plotted on a bar
graph for all treatments. The number on top of each bar graph represents the number of independent experiments conducted. (**p < 0.01 relative to
BIC+FSK?TTX treated sample).
Scale bars, 10 mm. See also Figure S5.
Cell 150, 207–221, July 6, 2012 ª2012 Elsevier Inc. 217
All primary, secondary antibodies, and protocols for immunoassays are
detailed in the Extended Experimental Procedures.
Plasmids and Neuronal Transfection
Transfections were done using calcium phosphate precipitation (Jiang and
Chen, 2006; Extended Experimental Procedures). Plasmids, cloning, and
PCR site-directed mutagenesis are described in Extended Experimental
Synaptosomes, PSD Fractionation, and 2D Gels
Synaptosomes and PSDs were prepared from adult rats (Sprague-Dawley)
and mice (C57/Bl6) as previously described by Jeffrey et al. (2009). Fluores-
cent signals from immunoblots were detected using the Odyssey Imaging
System(LI-COR).Detailed protocols for 2D gels are described in the Extended
Microscopes and Imaging
A Marianas spinning disc confocal microscope attached to a Photo-
metrics Evolve camera (Intelligent Imaging Innovation, Denver) was used
for live microscopy and single-plane quantification of nucleocytoplasmic
intensity. For high-resolution imaging of subcellular compartments, we
used a scanning confocal LSM 700 (Zeiss, Thornwood, NY, USA). For live-
referto Extended Experimental
Hippocampal Acute Slice and Organotypic Culture Studies
Organotypic Slice Cultures
Organotypic hippocampal slices were prepared as previously described by
Johnson and Buonomano (2007), and cLTP was induced as described in the
Extended Experimental Procedures.
Standard techniques approved by the UCLA IACUC were used to prepare
acute hippocampal slices from 8- to 16-week-old C57-Bl6 mice as previously
Figure 6. Activity Triggers Complex Changes in CRTC1 Phosphorylation
(A) Cultured hippocampal neurons were incubated with TTX (1 mM), bicuculline (BIC; 40 mM), or forskolin (FSK; 25 mM) in the presence or absence of CsA (5 mM).
After 10min,neuronal cultureswere lysed, separated by SDS-PAGE,and immunoblotted withantibodiesagainst TUJ1, CRTC1, or phosphorylated CRTC1-S151
(B) Cultured hippocampal neurons were stimulated as described in (A), and lysates were subjected to 2D gel electrophoresis and immunoblotted with antibodies
against CRTC1 (green) and TUJ1 (red).
(C) Neurons were transiently transfected with either full-length HA-tagged CRTC1 (HA-CRTC1) or CRTC1 bearing a point mutation converting serine 151 to
alanine (HA-CRTC1S151A). After 12 hr, transfected neurons were preincubated with either TTX (1 mM) for 1 hr or with bicuculline (40 mM) for 10 min before fixation
and immunostaining with antibodies to CRTC1 (green), HA (red), MAP2 (cyan), and Hoechst nuclear dye (blue). The nuclear-to-cytoplasmic ratio of HA
immunostaining was quantified (n.s., not significant). Scale bar, 10 mm.
See also Figure S6.
218 Cell 150, 207–221, July 6, 2012 ª2012 Elsevier Inc.
described by Delgado and O’dell (2005). Slices were maintained at 30?C in an
interface chamber (Fine Science Tools, Foster City, CA, USA) and recovered
for at least 2 hr before each experiment while being continuously perfused
(2–3 ml/min) with oxygenated (95% O2/5% CO2) ACSF (124 mM NaCl,
4.4 mM KCl, 25 mM Na2HCO3, 1 mM NaH2PO4, 1.2 mM MgSO4, 2 mM
CaCl2, and 10 mM glucose). Protocols for stimulation are described in
Extended Experimental Procedures.
Statistical significance was analyzed by one-way ANOVA and post hoc
Bonferroni’s multiple comparison test (GraphPad Prism, La Jolla, CA, USA)
unless otherwise noted.
figures, and one table and can be found with this article online at http://dx.doi.
We thank members of the Carew and K.C.M. lab for helpful discussions
and C. Alberini, K. Olofsdotter-Otis, V. Ho, C. Houser, and L. Zipursky for
critical reading of the manuscript. We thank M. Chin for advice on qPCR
experiments, M. DeSalvo for processing tissue samples, M. Haykinson for
Figure 7. CRTC1 Is Required for Activity-Dependent Induction of Specific CREB Target Genes
(A) Hippocampal neuron cultures (2–3 weeks) were incubated with Accell siRNA to CRTC1 (siCRTC1) or a nontargeted control (siNT). After 48 hr of siRNA
the siRNA-treated neurons were continuously maintained in TTX for an additional 30 min. Quantitative PCR was carried out to examine the concentrations of
activity-dependenttranscripts. Abar graph showing the relative fold change ofthesetranscripts betweenthesiCRTC1- and siNT-treated neuronswasplottedfor
both (i) TTX-treated and (ii) TTX-withdrawal conditions.
(B) Mouse hippocampal neurons (2–3 weeks) were incubated with Accell siRNA and treated as described above in (A). A third set of cultures was stimulated with
bicuculline (40mM; 10min). Neurons were lysed and analyzed by immunoblotting for CRTC1, TUJ1, and pCREB (S133). The relative concentration of pCREB was
plotted (n.s., not significant).
See also Figure S7.
Cell 150, 207–221, July 6, 2012 ª2012 Elsevier Inc. 219
slices. The work was supported by a NARSAD Young Investigator Award (to
T.H.C.), NIH R01 MH077022 (to K.C.M.), and R01 MH609197 (to T.J.O.).
Received: June 29, 2011
Revised: April 5, 2012
Accepted: May 2, 2012
Published: July 5, 2012
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