JuditEspan ˜a,1,2,3JorgeValero,1,2,3*AlfredoJ.Min ˜ano-Molina,1,2,3*RoserMasgrau,1,2ElsaMartín,1,2,3
CristinaGuardia-Laguarta,3,4AlbertoLleo ´,3,4LydiaGime ´nez-Llort,1,5Jose ´Rodríguez-Alvarez,1,2,3
1InstitutdeNeurocie `ncies,2DepartamentdeBioquímicaiBiologiaMolecular,3CentrodeInvestigacio ´nBiome ´dicaenRedEnfermedades
Activity-dependent gene expression mediating changes of synaptic efficacy is important for memory storage, but the mechanisms
underlying gene transcriptional changes in age-related memory disorders are poorly understood. In this study, we report that gene
active CRTC1 S151A and calcineurin mutants reverse the deficits on CRTC1 transcriptional activity in APPSw,Indneurons. Inhibition of
calcium influx by pharmacological blockade of L-type voltage-gated calcium channels (VGCCs), but not by blocking NMDA or AMPA
receptors, mimics the decrease on CRTC1 transcriptional activity observed in APPSw,Indneurons, whereas agonists of L-type VGCCs
reverse efficiently these deficits. Consistent with a role of CRTC1 on A?-induced synaptic and memory dysfunction, we demonstrate a
selective reduction of CRTC1-dependent genes related to memory (Bdnf, c-fos, and Nr4a2) coinciding with hippocampal-dependent
spatial memory deficits in APPSw,Indmice. These findings suggest that CRTC1 plays a key role in coupling synaptic activity to gene
to the balance between normal aging and age-related memory dis-
orders, including Alzheimer’s disease (AD) (Coleman and Yao,
2003; Berchtold et al., 2008). Synaptic dysfunction in AD is ap-
mechanisms underlying synaptic and memory dysfunction
caused by altered activity-dependent gene transcription in AD
are largely unknown. Understanding the molecular pathways
regulating gene expression profiles in memory disorders may al-
ery (Altar et al., 2009).
Activity-induced gene transcription mediates long-lasting
changes of synaptic efficacy essential for neuronal plasticity and
memory (Worley et al., 1993; Guzowski et al., 2001; Kandel,
2001). Thus, gene expression mediated by the transcription fac-
tor cAMP-response element binding protein (CREB) is essential
for synaptic plasticity and long-term memory (Bourtchuladze et
al., 1994; Won and Silva, 2008). CREB transcriptional activation
depends on calcium- and cAMP-dependent phosphorylation of
CREB at Ser133 (Sheng et al., 1991; Mayr and Montminy, 2001),
a process mediated by L-type voltage-gated calcium channels
(VGCCs) or glutamate ligand-gated receptors (NMDA and
AMPA) (Murphy et al., 1991; Cohen and Greenberg, 2008). In-
terestingly, altered cAMP/PKA-dependent CREB signaling has
aptic plasticity, memory, and synapse loss (Vitolo et al., 2002;
Gong et al., 2006; Smith et al., 2009).
Selective gene transcription by CREB depends on additional
events, including other phosphorylation sites and recruitment of
specific coactivators. The CREB-regulated transcription coacti-
cells, selective expression of CREB target genes in response to
cAMP and Ca2?signals, but not by stress stimuli, is achieved by
CB06/05/0042), Ministerio de Sanidad (FIS 04/0937 to C.A.S. and FIS 07/1137 to A.L.) and the 7th Framework
theServeideGeno `micaattheUniversitatAuto `nomadeBarcelona.
9402 • TheJournalofNeuroscience,July14,2010 • 30(28):9402–9410
tein (CBP)/p300 (Conkright et al., 2003; Ravnskjaer et al., 2007).
CRTC1, the most abundant isoform in neurons, mediates the
synergistic effect of calcium and cAMP signals on CREB-
dependent transcription and long-term potentiation (LTP)
involves its dephosphorylation by the calcium-dependent phos-
al., 2004; S. Li et al., 2009). Consistent with its role on CREB
activation, CRTC1 has been implicated in neuronal dendritic
growth, long-term synaptic plasticity, and glucose metabolism
(Zhou et al., 2006; Kova ´cs et al., 2007; Altarejos et al., 2008; S. Li
et al., 2009).
Whereas the function of CRTC1 on neuronal morphology
and plasticity is well established, its role on activity-dependent
gene transcription required for memory remains unknown. In
this study, we demonstrate that A? negatively regulates CRTC1
transgenic mice, resulting in a selective and differential disrup-
tion of CREB-dependent genes required for hippocampal-
Plasmids and antibodies. Mouse CRTC1-myc, Flag-CRTC2, CREB,
described (Janknecht et al., 1998; Conkright et al., 2003; Screaton et al.,
2004; Kova ´cs et al., 2007). Mouse calcineurin lacking functional CaM-
binding and autoinhibitory domains (?CnA) was cloned in the CMV-
Tag 4A plasmid (O’Keefe et al., 1992). pCRE-luc and TK-Renilla
plasmids were purchased from Stratagene and Promega. The CRTC1
S151A mutant was generated from pcDNA3-CRTC1-myc by standard
site-directed mutagenesis protocols (Stratagene) with the following for-
ward and reverse primers: 5?-GGAGGAGGACCAACGCTGACTCTG-
Rabbit phospho-Ser151 CRTC1 antibody was generated by immunizing
tide corresponding to mouse/human CRTC1 (amino acids 142-161).
sulfate precipitation and affinity purification (EZBiolab). The following
antibodies were used: APP/A? (6E10; 1:2000; Signet), CRTC1 (1:1000;
Cell Signaling Technology), CREB and pSer133 CREB (1:1000; Cell Sig-
naling Technology), Egr-1 (1:500; Santa Cruz Biotechnology), BDNF
(1:500; Alomone Labs), c-fos (1:500; Santa Cruz Biotechnology), lamin
B1 (1:500; Zymed), calcineurin (1:500; BD Transduction Labs), c-myc
(9E10; 1:1000; Santa Cruz Biotechnology), ?-actin (1:40,000; Abcam),
and ?-tubulin (1:20,000; Sigma-Aldrich).
APP transgenic mice and behavioral test. APPSw,Ind(line J9) transgenic
mice expressing mutant human APP695isoform harboring the FAD-
linked Swedish (K670N/M671L) and Indiana (V717F) mutations under
the neuronal PDGF? promoter have been previously described (Mucke
et al., 2000). Mice were age-matched littermate males obtained by cross-
ground). The Morris water maze was performed as previously described
(Gime ´nez-Llort et al., 2007; Espan ˜a et al., 2010). Experimenters of the
behavioral tests were blind to the genotypes of the mice. Animal proce-
dures were performed in accordance with institutional and national
guidelines following approval by the Animal Care and Ethical Commit-
tee (CEEAH) of the Universitat Auto `noma de Barcelona.
Primary neuronal culture and luciferase reporter assay. Primary neurons
were obtained from mouse embryos (E15.5) of heterozygous APPSw,Ind?
nontransgenic crossings. Neurons were dissociated and cultured in Neu-
at a density of 5 ? 104cells/cm2in 24-well or 35–60 mm dishes. For
luciferase assays, 7–15 d in vitro (DIV) neurons in 24-well dishes were
transfected for 24 h with pCRE-luc (0.5 ?g), TK-Renilla (0.25 ?g), and
vector or the indicated plasmids (0.5 ?g) by using LipofectAMINE 2000
shRNA lentiviral vectors (1–2 transducing units per cell). Neurons were
treated at 7 DIV with the indicated reagents before stimulation with
tions by using the Dual-Luciferase Assay System (Promega) in a Synergy
HT luminometer (Bio-Tek).
Lentiviral shRNA and ChIP. Complementary oligonucleotides for
mouse CRTC1 shRNA were as follows: Sh-CRTC1 forward:
TGTCACGCTGCttttt-3?; Sh-CRTC1 reverse: 5?-agctaaaaaGCAGCGTGA-
The scramble control oligonucleotides used were as follows: forward
TCCCAGCCttttt-3? and reverse: 5?-agctaaaaaGGCTGGGAATG-
were cloned into BglII/HindIII sites of the pSUPER.retro.puro plasmid
sites from pSUPER-Sh to generate the sequence H1-shRNA that was
inserted into pLVTHM vector. Lentiviral particles were generated in
HEK293T cells transfected with pLVTHM-Sh, pSPAX2, and pM2G
For chromatin immunoprecipitation (ChIP) assays, cortical neu-
rons (7 DIV) were treated with vehicle or FSK (20 ?M) plus KCl (30
mM) for 2 h. Cells were cross-linked with 1% formaldehyde, lysed in
ChIP buffer (25 mM HEPES, pH 8.0, 1.5 mM MgCl2, 10 mM KCl, 0.1%
NP-40, 1 mM DTT, and protease and phosphatase inhibitors) and
sonicated. Immunoprecipitations of DNA (2.5 ?g) were performed
overnight with anti-CRTC1 or irrelevant IgG (Cell Signaling Tech-
nology). PCR amplification was performed with specific primers for
CRE-containing promoters of the genes of interest (supplemental
Table 1S, available at www.jneurosci.org as supplemental material).
in 0.5 ml of cold-lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2
mM EDTA, 0.5% Triton X-100, 1% NP-40, 0.1% SDS, 1 mM Na3VO4, 50
inhibitors (Roche). For nuclear fractionation, primary neurons were in-
PMSF, supplemented with protease and phosphatase inhibitors). Ho-
mogenate was centrifuged (1500 ? g) at 4°C for 15 min. The pellet
(nuclei) was washed (twice) in buffer A and resuspended and sonicated
in lysis buffer (25 mM Tris HCl, pH 7.4, 150 mM NaCl, 1% NP-40, and
10% glycerol) supplemented with protease and phosphatase inhibitors.
Proteins were quantified using the BCA protein assay kit (Pierce) and
resolved on 8–12.5% SDS-PAGE gels. Proteins were visualized with the
enhanced chemiluminescence ECL kit (PerkinElmer) and quantified
with the ImageJ software within a linear range of detection for the ECL
reagent (Espan ˜a et al., 2010). Soluble A?-derived diffusible ligands
(ADDLs) were prepared freshly from synthetic A?1-42 peptides
(Bachem) as previously described (Klein, 2002). The same aggregation
were negative stained and examined in a JEOL JEM-2011 transmission
in conditioned medium using sensitive sandwich ELISA A?1-40and
A?1-42kits (Wako) (Guardia-Laguarta et al., 2009).
Calcineurin activity was determined with the calcineurin cellular ac-
tivity assay kit (Calbiochem). Briefly, mouse brain or cortical neurons
were homogenized in lysis buffer (25 mM Tris-HCl, pH 7.5, 0.5 mM
phate was eliminated using a desalting column, and equal amount of
?l of GREEN and fluorescence was measured at 620 nm using a micro-
titer plate reader.
Immunostaining of primary neurons. Cortical neurons (10 DIV) were
50 mM HEPES for 15 min. Cells were incubated in PBS containing 0.1 M
Espan ˜aetal.•CRTC1SignalinginanAlzheimer’sDiseaseModelJ.Neurosci.,July14,2010 • 30(28):9402–9410 • 9403
glycine, washed in PBS-Tween 0.1% (PBS-T), and permeabilized with
PBS-T plus 0.1% Triton X-100. Cells were blocked with PBS-T contain-
ing 0.5% normal goat serum, incubated overnight with mouse anti-
PSD-95 (1:50; BD Bioscience) and rabbit anti-synapsin I (1:500; Sigma)
antibodies, and detected with the Alexa Fluor 488 or 594 secondary an-
tibodies (1:500) and Hoechst 33258 (Invitrogen). Images from control
confocal laser microscopy (Leica TCS SP2 AOBS; Carl Zeiss). For quan-
titative analyses, acquired neurite images (n ? 15 per genotype) were
were defined as being 0.5–2 ?m in length, twofold to threefold more
intense than background staining, and stained for synapsin, PSD-95, or
Calcium imaging. Primary cortical neurons grown onto poly-lysine-
2/AM (4 ?M) for 1 h. Coverslips were washed with Krebs buffer
containing (in mM) 119 NaCl, 4.75 KCl, 5 NaHCO3, 1.2 MgSO4, 1.18
KH2PO4, 1.3 CaCl2, 20 HEPES, and 10 glucose, pH 7.4, and mounted in
a static chamber at 37°C on an inverted Nikon TE2000U microscope.
tor (Cairn Research Limited), and emission light collected at 510 nm
every 4 s. Images were acquired by using a 12 bit-CCD ERG ORCA
sal Imaging). When appropriated, cells were treated with KCl (30 mM)
and forskolin (20 ?M). n ? 15 cells/genotype (n ? 3 embryos) were
analyzed in each experiment.
Real-time RT-PCR. Total RNA was isolated from mouse primary cor-
Purified RNA was reverse transcribed using the SuperScript II Reverse
of Oligo(dT) primers, 0.5 mM dNTP, 0.45 mM DTT, RNaseOut (10 U),
and SuperScript II Reverse Transcriptase (200 U; Invitrogen) was incu-
bated at 25°C for 10 min, 42°C for 60 min, and 72°C for 10 min. Quan-
titative RT-PCR of a reaction mix containing cDNA (1 ?l), primer pairs
(supplemental Table 1S, available at www.jneurosci.org as supplemental
material), and the QuantiMix EASY SYG KIT mix (10 ?l; Biotools) was
performed in an ABI PRISM 7900 Sequence Detector (Applied Biosys-
tems). Data analysis was performed by the comparative ??Ct method
using the SDS 2.1 software and normalizing to GAPDH.
Statistical analysis. Statistical analysis was performed using one-way
using two-way ANOVA with repeated measures and Scheffe ´’s S test for
with p ? 0.05 were considered significant.
To evaluate the possible role of CREB signaling on A?-induced
established primary neurons from an ?-amyloid precursor pro-
tein (APP) transgenic mouse (APPSw,Ind) that develops age-
2000; Espan ˜a et al., 2010). Cortical neurons from APPSw,Indem-
bryos expressed human APP (approximately twofold) and re-
leased soluble A?1-40and A?1-42peptides without causing gross
morphological synaptic changes (supplemental Fig. 1S, available
at www.jneurosci.org as supplemental material). Confocal mi-
ber of presynaptic (synapsin), postsynaptic (PSD-95), or active
(synapsin/PSD-95) synapses (supplemental Fig. 2S, available at
www.jneurosci.org as supplemental material). Since synaptic ac-
tivity induces efficient expression of immediate-early genes in
cortical neurons over the course of 1–3 weeks (Murphy et al.,
1991), we performed CREB transcriptional analysis in neu-
rons at 7–15 DIV in conditions mimicking the effects of neu-
ronal activity, such as increasing intracellular Ca2?by
depolarizing concentrations of KCl (30 mM) or cAMP with the
adenylate cyclase activator forskolin (FSK) (Greer and Green-
berg, 2008). Treatment of control neurons with FSK or KCl
resulted in ?2- and ?7-fold increase on CRE-luciferase activity,
respectively, whereas their combination induced a synergistic ef-
transcription was unchanged by FSK but was significantly re-
duced by KCl (?25%) or KCl plus FSK (?50%) in cortical and
hippocampal APPSw,Indneurons (Fig. 1A). Inhibitors of synaptic
activity (tetrodotoxin TTX) or calcineurin (FK-506 and cyclo-
by Ca2?and cAMP signals (Fig. 1A).
Consistent with the idea that CRTC mediates the synergistic
effect of cAMP and Ca2?on CREB-dependent transcription
(Screaton et al., 2004), we found that CRTC1, CRTC2, CBP, or
p300, but not the CREB R314A mutant lacking the CRTC bind-
ing domain (Screaton et al., 2004), potentiated and reversed
CRE-transcriptional deficits in APPSw,Indneurons (Fig. 1B).
These results suggested a role of CRTC on altered activity-
induced CRE-transcription in APPSw,Indneurons. We then fo-
cused on CRTC1, the most abundant CRTC isoform in neurons
and brain (Kova ´cs et al., 2007; Altarejos et al., 2008). We gener-
ated lentiviral vectors expressing CRTC1 shRNA that decreased
significantly CRTC1 (62–75%) and CRE-mediated transcription
induced by cAMP and Ca2?signals (?80%) (Fig. 1C). To study
the biological significance of CREB transcriptional deficits, we
CRTC1 by shRNA and gene ChIP analyses demonstrated that
CRTC1 is recruited to and activates CRE-containing promoters
of several genes, including c-fos, Bdnf IV, and Nr4a2 but not
Cyr61 (Fig. 1D,E). Interestingly, induced expression of endoge-
nous CRTC1-dependent genes related to synaptic plasticity and
memory such as c-fos, Bdnf IV, and Nr4a2 (?100- to 1000-fold),
but not Cyr61 (?15-fold), a CREB target gene related to prolif-
2007), was significantly decreased in APPSw,Indcortical neurons
CRTC1 in APPSw,Indneurons, we used pharmacological and ge-
netic approaches previously shown to reduce A? levels (Saura et
al., 2005; Oddo et al., 2006). Decreasing A? with the ?-secretase
reversed significantly CRE-transcriptional deficits in APPSw,Ind
neurons (Fig. 2A; supplemental Fig. 1S, available at www.
jneurosci.org as supplemental material). Surprisingly, affecting
only extracellular A? by treatment with Ab20.1 reversed only
tivation of PS1/?-secretase in APPSw,Indneurons resulted in nor-
mal levels of CRE-transcriptional activity (data not shown). By
contrast, media from APPSw,Indneurons or soluble globular syn-
thetic A?1-42oligomers (ADDLs) containing dimers, trimers,
hexamers, and dodecamers at concentrations not affecting neu-
ron morphology or viability (1–20 ?M) (Klein, 2002), but not
A?1-42monomers or A?42-1peptides submitted to the aggrega-
tion protocol, reduced significantly CREB-dependent transcrip-
tion in a dose-dependent manner (Fig. 2B,C). These results
suggested that A? negatively affects activity-induced CRTC1-
dependent transcription in primary neurons.
The inhibitory effect of calcineurin inhibitors, the CREB R314R
9404 • J.Neurosci.,July14,2010 • 30(28):9402–9410 Espan ˜aetal.•CRTC1SignalinginanAlzheimer’sDiseaseModel
prompted us to examine the role of A? on calcineurin-mediated
CRTC1 activation. Surprisingly, whereas both staurosporine
(STS), at doses reported to inhibit SIK and promoting CRTC2
activation [10 nM (Ravnskjaer et al., 2007)], and the active cal-
cineurin mutant ?CnA (O’Keefe et al., 1992) potentiated
CRTC1-dependent transcription, only expression of ?CnA re-
versed efficiently transcriptional deficits in APPSw,Indneurons
(Fig. 3A). Biochemical and quantitative analyses revealed that
FSK/KCl-induced CRTC1 dephosphorylation was significantly
reduced in APPSw,Indneurons in total and nuclear lysates ( p ?
0.01). Levels of total CRTC1 (WT: 1.0 ? 0.4 vs APPSw,Ind: 0.86 ?
phosphorylated CREB (WT: 1.8 ? 0.6 vs APPSw,Ind: 2.0 ? 0.3-
endogenous CRTC1 is efficiently downregulated by CRTC1 shRNA in cortical neurons. CRE-
regulated by CRTC1 shRNA in response to KCl/FSK treatment. Bottom, Quantitative real-time
FSK/KCl in APPSw,Indneurons. Values of each gene are normalized to GAPDH and represent
response to FSK/KCl. IgG indicates immunoprecipitation with an irrelevant antibody. Input
Espan ˜aetal.•CRTC1SignalinginanAlzheimer’sDiseaseModel J.Neurosci.,July14,2010 • 30(28):9402–9410 • 9405
fold) were unchanged in APPSw,Indneu-
rons (Fig. 3B). To study the biological
Ser151, a phosphorylation site equivalent
to CRTC2 Ser 171 (Altarejos et al., 2008),
we developed a phosphoSer151-specific
CRTC1 antiserum that recognized the
endogenous and overexpressed phos-
phorylated CRTC1 but not a phospho-
rylation-defective CRTC1 S151A mutant
or CRTC2 (Fig. 3C; data not shown). No-
tably, CRTC1 phosphorylation at Ser151
was significantly increased (?2-fold) in
the hippocampus of APPSw,Indmice (Fig.
CRTC1 S151A mutant enhanced and re-
versed CREB transcriptional deficits in
APPSwe,Indneurons (Fig. 3E). These results
strongly suggested a deficit on calci-
tion in neurons and brain of APPSw,Ind
Because calcineurin requires Ca2?for its
activation, we next examined the effect of
Ca2?signaling disruption on CRTC1-
mediated transcription. Blockers of intra-
cellular Ca2?(BAPTA), Ca2?influx
doplasmic reticulum (thapsigargin) sig-
transcription in cortical neurons (sup-
plemental Fig. 3S, available at www.
jneurosci.org as supplemental material).
Indeed, calcineurin activity was signifi-
cantly reduced in hippocampal (? 25%)
and cortical (?40%) neurons and adult
brain (?47%) from APPSw,Indmice (Fig. 4A). Western blotting
binding catalytic and Ca2?-binding subunits in APPSw,Indneu-
rons (Fig.4B). We then
concentration changes elicited by depolarization and cAMP,
which are mediated by Ca2?influx from L-type voltage-gated
calcium channels (VGCCs) and Ca2?mobilization from intra-
cellular stores. Accordingly, Ca2?imaging experiments showed
that the amplitude of Ca2?changes elicited by FSK/KCl treat-
ment was significantly reduced in APPSw,Indneurons (Fig. 4C).
Similar percentage of control and APPSw,Indneurons responded to
L-type VGCCs greatly contribute to Ca2?-induced gene ex-
pression in hippocampal neurons (Murphy et al., 1991; Mintz et
blockers of postsynaptic L-type (verapamil and nimodipine) or
presynaptic N/P/Q-type (?-conotoxin) VGCCs, but not AMPA
(CNQX) or NMDA (MK-801) antagonists, reduced and oc-
trol and APPSw,Indneurons, respectively (Fig. 4D; supplemental
Fig. 3S, available at www.jneurosci.org as supplementalmaterial).
Accordingly, the specific L-type Ca2?channel agonists BayK-8644,
tional deficits in APPSw,Indneurons (Fig. 4E). Altogether, these re-
sults demonstrated that deficient calcium influx through L-type
VGCCs was directly involved in disruption of CRTC1-dependent
Having seen the critical role of A? on CRTC1-mediated tran-
scription, we finally analyzed its effect on memory deficits in
APPSw,Indtransgenic mice at an age (6 months) coinciding with
initial A?40/A?42 accumulation (Mucke et al., 2000; Espan ˜a et
al., 2010) (data not shown). We used the Morris water maze, a
spatial memory task that induces expression of immediate early
genes 0.5–1 h after training (Guzowski et al., 2001). Six-month-
old APPSw,Indmice required significantly longer latencies and
distances to locate the platform during training (two-way
ANOVA; latencies: genotype effect, F(1,50)? 19.9; day effect,
F(4,50)? 31.6; p ? 0.0001). In the probe trial, APPSw,Indmice
spent significantly less time searching and crossing the target
platform than controls (genotype effect: F(1,40)? 5.6; quadrant
effect: F(3,40)? 6.1, p ? 0.002) (Fig. 5A and data not shown).
Quantitative real-time RT-PCR analysis revealed a selective re-
duction of CREB target genes regulated by CRTC1 related to
CRTC1, CREB, and pCREB (Ser133) in total and nuclear lysates from control (WT) and APPSw,Indneurons (n ? 3). The lower
migrating band corresponding to dephosphorylated CRTC1 (top blot, upper graph) and nuclear CRTC1 levels (bottom blot and
9406 • J.Neurosci.,July14,2010 • 30(28):9402–9410 Espan ˜aetal.•CRTC1SignalinginanAlzheimer’sDiseaseModel
memory (c-fos, Bdnf, and Nr4a2) but not to stress (Rgs2) (Fig. 1)
(Ravnskjaer et al., 2007) and unchanged CREB genes regulated in-
dependently of CRTC1 involved in memory processing (Egr1), cell
of trained APPSw,Indmice (Fig. 5B,C). Reduced expression of mR-
NAs was associated with a significant decreased of c-fos and Bdnf
of CRTC1-dependent genes coinciding with A? accumulation
and hippocampal-dependent spatial memory deficits in APPSw,Ind
Gene expression changes in the brain have been suggested to
underlie synaptic and cognitive dysfunction during normal and
pathological aging (Coleman and Yao, 2003; Berchtold et al.,
2008). The molecular mechanisms underlying gene expression
study, we identified the transcriptional coactivator CRTC1 as
mediating the effect of A? on disrupting synaptic coupling to
activation of genes required for neuronal plasticity and memory.
The temporal coincidence of deregulated CRTC1-dependent
strongly argues for a role of CRTC1 on mediating memory pro-
cessing in normal and pathological conditions.
The transcription factor CREB is a key contributor to cAMP-
and calcium-dependent gene transcription during synaptic de-
Silva, 2008). CREB signaling requires phosphorylation of CREB
on Ser133 by cAMP- and Ca2?/calmodulin-dependent kinases
(Gonzalez and Montminy, 1989; Dash et al., 1991; Sheng et al.,
1991). However, CREB phosphorylation is not sufficient to acti-
vate gene transcription (Bito et al., 1996; Zhang et al., 2005),
requiring the coactivators CBP, p300, and CRTC (Chrivia et al.,
1993; Conkright et al., 2003; Ravnskjaer et al., 2007). Our results
showing similar increase of CREB phosphorylation by calcium/
tent with previous reports demonstrating unchanged CREB
phosphorylation by A?42 in basal or FSK-stimulated mature
By contrast, CREB phosphorylation is decreased in AD brain
(Yamamoto-Sasaki et al., 1999) and oligomeric A?42 suppresses
NMDA- and depolarization-induced CREB phosphorylation in
immature neurons (Tong et al., 2001; Ma et al., 2007), an effect
that may be due to cAMP/PKA signaling deregulation (Vitolo et
al., 2002). Indeed, it was previously shown that A? alters hip-
pocampal synaptic plasticity, memory, and synapse morphology
tiating this pathway reverses those deficits (Gong et al., 2004,
2006; Smith et al., 2009).
gene expression in response to neuronal activity (Belfield et al.,
2006), but they act synergistically on CREB signaling by activat-
ing the transcriptional coactivator CRTC (Screaton et al., 2004;
Kova ´cs et al., 2007). Consistently, we found deregulation of
CRTC1-dependent CREB transcription and reduced Ca2?re-
SEM of three independent cultures per genotype (n ? 15 cells per culture). D, L-type VGCC blockers nimodipine (5 ?M) and verapamil (100 ?M) mimic and occlude the effect of A? on
Espan ˜aetal.•CRTC1SignalinginanAlzheimer’sDiseaseModel J.Neurosci.,July14,2010 • 30(28):9402–9410 • 9407
sponses by naturally secreted A? or synthetic A? oligomers.
These transcriptional deficits are likely attributable to the direct
effect of A? on CRTC1 because they were prevented by pharma-
CRTC1 or CRTC1 S151A but not CREB. Surprisingly, treatment
of neurons with Ab20.1 antibody reversed only partially CRE-
tracellular A? could contribute to the CRTC1-transcriptional
than CREB account for the observed activity-dependent tran-
scriptional deficits in our AD neuronal model.
derlying gene expression changes in AD. Activity-dependent gene
expression mediated by CREB in excitatory neurons depends on
calcium influx through L-type VSCCs or either NMDA or AMPA
glutamate receptors (Greer and Greenberg, 2008). Though L-type
VGCCs make a minor contribution to synaptic-induced calcium
current, they play a critical role in coupling synaptic stimulation to
activation of nuclear gene expression (Murphy et al., 1991; Greer
and Greenberg, 2008). In agreement with this view, we found that
depletion of intracellular calcium and blockers of VGCCs, but not
NMDA or AMPA receptor antagonists, reduced significantly
CRTC1 transcriptional activity in control neurons, whereas they
Junb, Egr-1, and Fosb are primarily activated by calcium entry
the Cav1.2 channel suggest a role of L-type Ca2?channels on
NMDAR-independent hippocampal LTP and CREB transcription
(Moosmang et al., 2005). Because A? depresses excitatory synaptic
transmission through AMPA and NMDA receptors (Snyder et al.,
A? modulates differentially glutamatergic signaling depending on
The finding that agonists of L-type Ca2?channels reversed
the CREB transcriptional deficits in APPSw,Indcortical neurons
on the A?-induced CRTC1 transcriptional deficits. Indeed, cal-
cium imaging analysis demonstrated decreased intracellular
Ca2?mobilization in response to depolarization and cAMP sig-
Ca2?responses, A? reduces P/Q-type calcium currents and
mice (Busche et al., 2008; Nimmrich et al., 2008). One of the
consequences of reduced calcium responses by A? may be an
et al., 2007), which in turn may result in decreased L-type Ca2?
channel function (Norris et al., 2002; Tandan et al., 2009). Con-
sistent with an essential role of calcineurin on depolarization-
induced CREB-dependent transcription and CRTC1 function
activity and CRTC1 dephosphorylation induced by calcium and
cAMP signals were fully blocked by calcineurin inhibitors,
whereas the active ?CnA mutant efficiently reversed the tran-
scriptional deficits in APPSw,Indneurons. In support of a role of
altered dephosphorylation of CRTC1 on CRE-transcriptional
deficits, we found that SIK inhibition was unable to reverse effi-
ficient PKA-mediated L-type Ca2?channel function, cannot be
plays an essential role in plasticity mechanisms required for
memory processing (Guzowski et al., 2005). Neuronal activity
and memory training induce expression of Bdnf, c-fos, Junb,
impaired APPSw,Indmice. A, APPSw,Indtransgenic mice display learning and spatial memory
but they required significantly longer latencies to locate the platform (two-way ANOVA;
latencies: genotype effect, F(1,50)? 19.9; day effect, F(4,50)? 31.6; p ? 0.0001). In the
target quadrant (TQ) platform location than nontransgenic controls. Data represent the
during the probe trial. OP, Opposite platform; AR, adjacent right platform; AL, adjacent left
platform. B, Quantitative analysis of hippocampal mRNA of CREB target genes by real-time
controls. Bdnf refers to Bdnf IV. D, Western blot images showing reduction of c-fos and
Reduced CRTC1-dependent CREB target genes in the hippocampus of cognitive
9408 • J.Neurosci.,July14,2010 • 30(28):9402–9410Espan ˜aetal.•CRTC1SignalinginanAlzheimer’sDiseaseModel
Egr-1, and Fosb (Murphy et al., 1991; Worley et al., 1993;
Guzowski et al., 2001). Notably, we found that deregulation on
cided with the first long-term spatial memory deficits in
APPSw,Indmice (Espan ˜a et al., 2010), suggesting that these events
are tightly linked early in the disease process. Notably, reduced
c-fos levels were recently associated with learning and memory
deficits in APP transgenic mice (Palop et al., 2003; Dewachter et
al., 2009), whereas BDNF is decreased in brains of AD patients
and transgenic mice (Phillips et al., 1991; Dickey et al., 2003;
Palop et al., 2003). In this regard, Bdnf IV, which is induced by
calcium influx during neuronal activity and is downregulated by
A? (Tong et al., 2001; Garzon and Fahnestock, 2007), is particu-
larly important. Our finding that disruption of Bdnf IV and c-fos
expression is mediated by deregulation of CRTC1 in APP trans-
genic mice provides the first reported molecular mechanism un-
derlying deregulation of c-fos and BDNF signaling in AD.
In conclusion, our finding that A? disrupts expression of
CRTC1 target genes essential for memory processing provides a
potential mechanism contributing to cognitive decline in AD.
These results may have important therapeutic implications in
AD. Indeed, reduced levels of BDNF in CSF were recently asso-
ciated with age-related cognitive decline (G. Li et al., 2009),
whereas BDNF exerts substantial protective effects on neuronal
survival and memory circuits in rodent and primate models of
AD (Nagahara et al., 2009). Similarly, neural stem cells trans-
planted in the hippocampus of 3xTg-AD mice enhance synaptic
density and improve cognitive function through BDNF (Blurton-
Jones et al., 2009). Importantly, agents that activate the PKA/
CREB signaling pathway, such as rolipram, ameliorate hippo-
campal-dependent memory deficits and synapse loss in APP
understanding the molecular mechanisms regulating CRTC1-
dependent signaling and gene responses to therapeutic drugs may
provide new targets for memory enhancement in cognitive
Altar CA, Vawter MP, Ginsberg SD (2009) Target identification for CNS
diseases by transcriptional profiling. Neuropsychopharmacology 34:
PE, Montminy M (2008) The Creb1 coactivator Crtc1 is required for
energy balance and fertility. Nat Med 14:1112–1117.
Belfield JL, Whittaker C, Cader MZ, Chawla S (2006) Differential effects of
Ca2?and cAMP on transcription mediated by MEF2D and cAMP-
response element-binding protein in hippocampal neurons. J Biol Chem
Berchtold NC, Cribbs DH, Coleman PD, Rogers J, Head E, Kim R, Beach T,
Miller C, Troncoso J, Trojanowski JQ, Zielke HR, Cotman CW (2008)
Gene expression changes in the course of normal brain aging are sexually
dimorphic. Proc Natl Acad Sci U S A 105:15605–15610.
BitoH,DeisserothK,TsienRW (1996) CREBphosphorylationanddephos-
pocampal gene expression. Cell 87:1203–1214.
Bittinger MA, McWhinnie E, Meltzer J, Iourgenko V, Latario B, Liu X, Chen
CH, Song C, Garza D, Labow M (2004) Activation of cAMP response
element-mediated gene expression by regulated nuclear transport of
TORC proteins. Curr Biol 14:2156–2161.
Blurton-Jones M, Kitazawa M, Martinez-Coria H, Castello NA, Mu ¨ller FJ,
Loring JF, Yamasaki TR, Poon WW, Green KN, LaFerla FM (2009)
Neural stem cells improve cognition via BDNF in a transgenic model of
Alzheimer disease. Proc Natl Acad Sci U S A 106:13594–13599.
Bourtchuladze R, Frenguelli B, Blendy J, Cioffi D, Schutz G, Silva AJ (1994)
Deficient long-term memory in mice with a targeted mutation of the
c-AMP-responsive element binding protein. Cell 79:59–68.
Busche MA, Eichhoff G, Adelsberger H, Abramowski D, Wiederhold KH,
Haass C, Staufenbiel M, Konnerth A, Garaschuk O (2008) Clusters of
hyperactive neurons near amyloid plaques in a mouse model of Alzhei-
mer’s disease. Science 321:1686–1689.
Celsi F, Svedberg M, Unger C, Cotman CW, Carrì MT, Ottersen OP, Nord-
bergA,TorpR (2007) ?-Amyloidcausesdownregulationofcalcineurin
in neurons through induction of oxidative stress. Neurobiol Dis
(1993) Phosphorylated CREB binds specifically to the nuclear protein
CBP. Nature 365:855–859.
Cohen S, Greenberg ME (2008) Communication between the synapse and
the nucleus in neuronal development, plasticity, and disease. Annu Rev
Cell Dev Biol 24:183–209.
ColemanPD,YaoPJ (2003) SynapticslaughterinAlzheimer’sdisease.Neu-
robiol Aging 24:1023–1027.
Conkright MD, Canettieri G, Screaton R, Guzman E, Miraglia L, Hogenesch
JB, Montminy M (2003) TORCs: transducers of regulated CREB activ-
ity. Mol Cell 12:413–423.
Dash PK, Karl KA, Colicos MA, Prywes R, Kandel ER (1991) cAMP re-
as cAMP-dependent protein kinase. Proc Natl Acad Sci U S A
Davare MA, Hell JW (2003) Increased phosphorylation of the neuronal
L-type Ca2?channel Ca(v)1.2 during aging. Proc Natl Acad Sci U S A
Dewachter I, Filipkowski RK, Priller C, Ris L, Neyton J, Croes S, Terwel D,
Gysemans M, Devijver H, Borghgraef P, Godaux E, Kaczmarek L, Herms
J, Van Leuven F (2009) Deregulation of NMDA-receptor function and
down-stream signaling in APP[V717I] transgenic mice. Neurobiol Aging
Dickey CA, Loring JF, Montgomery J, Gordon MN, Eastman PS, Morgan D
in amyloid precursor protein ? presenilin-1 transgenic mice. J Neurosci
Espan ˜a J, Gime ´nez-Llort L, Valero J, Min ˜ano A, Ra ´bano A, Rodriguez-
Alvarez J, LaFerla FM, Saura CA (2010) Intraneuronal ?-amyloid accu-
mulation in the amygdala enhances fear and anxiety in Alzheimer’s
disease transgenic mice. Biol Psychiatry 67:513–521.
GarzonDJ,FahnestockM (2007) Oligomericamyloiddecreasesbasallevels
of brain-derived neurotrophic factor (BDNF) mRNA via specific down-
regulation of BDNF transcripts IV and V in differentiated human neuro-
blastoma cells. J Neurosci 27:2628–2635.
Ghosh A, Carnahan J, Greenberg ME (1994) Requirement for BDNF in
activity-dependent survival of cortical neurons. Science 263:1618–1623.
Gime ´nez-Llort L, Bla ´zquez G, Can ˜ete T, Johansson B, Oddo S, Toben ˜a A,
LaFerla FM, Ferna ´ndez-Teruel A (2007) Modeling behavioral and neu-
ronal symptoms of Alzheimer’s disease in mice: a role for intraneuronal
amyloid. Neurosci Biobehav Rev 31:125–147.
Gong B, Vitolo OV, Trinchese F, Liu S, Shelanski M, Arancio O (2004)
Gong B, Cao Z, Zheng P, Vitolo OV, Liu S, Staniszewski A, Moolman D,
Zhang H, Shelanski M, Arancio O (2006) Ubiquitin hydrolase Uch-L1
memory. Cell 126:775–788.
Gonzalez GA, Montminy MR (1989) Cyclic AMP stimulates somatostatin
gene transcription by phosphorylation of CREB at serine 133. Cell
Greer PL, Greenberg ME (2008) From synapse to nucleus: calcium-
dependent gene transcription in the control of synapse development and
function. Neuron 59:846–860.
Guardia-Laguarta C, Coma M, Pera M, Clarimo ´n J, Sereno L, Agullo ´ JM,
Go ´mez-Isla T, Lleo ´ A (2009) Mild cholesterol depletion reduces
amyloid-? production by impairing APP trafficking to the cell surface.
J Neurochem 110:220–230.
Guzowski JF, Setlow B, Wagner EK, McGaugh JL (2001) Experience-
dependent gene expression in the rat hippocampus after spatial learning:
a comparison of the immediate-early genes Arc, c-fos, and zif268. J Neu-
Guzowski JF, Timlin JA, Roysam B, McNaughton BL, Worley PF, Barnes CA
Espan ˜aetal.•CRTC1SignalinginanAlzheimer’sDiseaseModelJ.Neurosci.,July14,2010 • 30(28):9402–9410 • 9409
(2005) Mapping behaviorally relevant neural circuits with immediate- Download full-text
early gene expression. Curr Opin Neurobiol 15:599–606.
Hsieh H, Boehm J, Sato C, Iwatsubo T, Tomita T, Sisodia S, Malinow R
(2006) AMPAR removal underlies A?-induced synaptic depression and
dendritic spine loss. Neuron 52:831–843.
Iourgenko V, Zhang W, Mickanin C, Daly I, Jiang C, Hexham JM, Orth AP,
Miraglia L, Meltzer J, Garza D, Chirn GW, McWhinnie E, Cohen D,
Skelton J, Terry R, Yu Y, Bodian D, Buxton FP, Zhu J, Song C, et al.
(2003) Identification of a family of cAMP response element-binding
protein coactivators by genome-scale functional analysis in mammalian
cells. Proc Natl Acad Sci U S A 100:12147–12152.
Janknecht R, Wells NJ, Hunter T (1998) TGF-beta-stimulated cooperation
of smad proteins with the coactivators CBP/p300. Genes Dev
Kandel ER (2001) The molecular biology of memory storage: a dialogue
between genes and synapses. Science 294:1030–1038.
Kingsbury TJ, Bambrick LL, Roby CD, Krueger BK (2007) Calcineurin ac-
scription in cortical neurons. J Neurochem 103:761–770.
Klein WL (2002) A? toxicity in Alzheimer’s disease: globular oligomers
(ADDLs) as new vaccine and drug targets. Neurochem Int 41:345–352.
Kova ´cs KA, Steullet P, Steinmann M, Do KQ, Magistretti PJ, Halfon O, Car-
dinauxJR (2007) TORC1isacalcium-andcAMP-sensitivecoincidence
Acad Sci U S A 104:4700–4705.
Li G, Peskind ER, Millard SP, Chi P, Sokal I, Yu CE, Bekris LM, Raskind MA,
Galasko DR, Montine TJ (2009) Cerebrospinal fluid concentration of
brain-derived neurotrophic factor and cognitive function in non-
demented subjects. PLoS ONE 4:e5424.
Li S, Zhang C, Takemori H, Zhou Y, Xiong ZQ (2009) TORC1 regulates
of developing cortical neurons. J Neurosci 29:2334–2343.
Lian Q, Ladner CJ, Magnuson D, Lee JM (2001) Selective changes of cal-
cortex. Exp Neurol 167:158–165.
Ma QL, Harris-White ME, Ubeda OJ, Simmons M, Beech W, Lim GP, Teter
B, Frautschy SA, Cole GM (2007) Evidence of Abeta- and transgene-
dependent defects in ERK-CREB signaling in Alzheimer’s models. J Neu-
Mayr B, Montminy M (2001) Transcriptional regulation by the
phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol
Mintz IM, Adams ME, Bean BP (1992) P-type calcium channels in rat cen-
tral and peripheral neurons. Neuron 9:85–95.
Moosmang S, Haider N, Klugbauer N, Adelsberger H, Langwieser N, Mu ¨ller
J, Stiess M, Marais E, Schulla V, Lacinova L, Goebbels S, Nave KA, Storm
DR,HofmannF,KleppischT (2005) RoleofhippocampalCav1.2Ca2?
channels in NMDA receptor-independent synaptic plasticity and spatial
memory. J Neurosci 25:9883–9892.
Mucke L, Masliah E, Yu GQ, Mallory M, Rockenstein EM, Tatsuno G, Hu K,
Kholodenko D, Johnson-Wood K, McConlogue L (2000) High-level
neuronal expression of A?1-42in wild-type human amyloid protein pre-
Murphy TH, Worley PF, Baraban JM (1991) L-type voltage-sensitive cal-
cium channels mediate synaptic activation of immediate early genes.
Geschwind D, Masliah E, Chiba AA, Tuszynski MH (2009) Neuropro-
models of Alzheimer’s disease. Nat Med 15:331–337.
Hillen H, Gross G, Ebert U, Bruehl C (2008) Amyloid ? oligomers
of P/Q-type calcium currents. J Neurosci 28:788–797.
Norris CM, Blalock EM, Chen KC, Porter NM, Landfield PW (2002) Cal-
cineurin enhances L-type Ca(2?) channel activity in hippocampal neu-
rons: increased effect with age in culture. Neuroscience 110:213–225.
Oddo S, Vasilevko V, Caccamo A, Kitazawa M, Cribbs DH, LaFerla FM
(2006) Reduction of soluble abeta and tau, but not soluble abeta alone,
J Biol Chem 281:39413–39423.
O’KeefeSJ,TamuraJ,KincaidRL,TocciMJ,O’NeillEA (1992) FK-506-and
CsA-sensitive activation of a interleukin-2 promoter by calcineurin.
Palop JJ, Jones B, Kekonius L, Chin J, Yu GQ, Raber J, Masliah E, Mucke L
gyrus is tightly linked to Alzheimer’s disease-related cognitive deficits.
Proc Natl Acad Sci U S A 100:9572–9577.
Phillips HS, Hains JM, Armanini M, Laramee GR, Johnson SA, Winslow JW
(1991) BDNF mRNA is decreased in the hippocampus of individuals
with Alzheimer’s disease. Neuron 7:695–702.
Puzzo D, Privitera L, Leznik E, Fa ` M, Staniszewski A, Palmeri A, Arancio O
memory in hippocampus. J Neurosci 28:14537–14545.
Ravnskjaer K, Kester H, Liu Y, Zhang X, Lee D, Yates JR 3rd, Montminy M
(2007) Cooperative interactions between CBP and TORC2 confer selec-
tivity to CREB target gene expression. EMBO J 26:2880–2889.
Kirkwood A, Morris RG, Shen J (2005) Conditional inactivation of
presenilin-1 prevents amyloid accumulation and temporarily rescues
contextual and spatial working memory impairments in amyloid precur-
sor protein transgenic mice. J Neurosci 25:6755–6764.
Niessen S, Yates JR 3rd, Takemori H, Okamoto M, Montminy M (2004)
The CREB coactivator TORC2 functions as a calcium- and cAMP sensitive
Selkoe DJ (2002) Alzheimer’s disease is a synaptic failure. Science 298:
Sheng M, Thompson MA, Greenberg ME (1991) CREB: a Ca2?-regulated
transcription factor phosphorylated by calmodulin-dependent kinases.
Smith DL, Pozueta J, Gong B, Arancio O, Shelanski M (2009) Reversal of
long-term dendritic spine alterations in Alzheimer disease models. Proc
Natl Acad Sci U S A 106:16877–16882.
MW, Lombroso PJ, Gouras GK, Greengard P (2005) Regulation of
NMDA receptor trafficking by amyloid-?. Nat Neurosci 8:1051–1058.
Tandan S, Wang Y, Wang TT, Jiang N, Hall DD, Hell JW, Luo X, Rothermel
BA, Hill JA (2009) Physical and functional interaction between cal-
cineurin and the cardiac L-type Ca2?channel. Circ Res 105:51–60.
Tong L, Thornton PL, Balazs R, Cotman CW (2001) ?-amyloid-(1-42) im-
pairs activity-dependent cAMP-response element-binding protein sig-
naling in neurons at concentrations in which cell survival is not
compromised. J Biol Chem 276:17301–17306.
Vitolo OV, Sant’Angelo A, Costanzo V, Battaglia F, Arancio O, Shelanski M
(2002) Amyloid ?-peptide inhibition of the PKA/CREB pathway and
long-term potentiation: reversibility by drugs that enhance cAMP signal-
ing. Proc Natl Acad Sci U S A 99:13217–13221.
TakasuMA,TaoX,GreenbergME (2001) Calciumregulationofneuro-
nal gene expression. Proc Natl Acad Sci U S A 98:11024–11031.
WonJ,SilvaAJ (2008) Molecularandcellularmechanismsofmemoryallo-
cation in neuronetworks. Neurobiol Learn Mem 89:285–292.
Worley PF, Bhat RV, Baraban JM, Erickson CA, McNaughton BL, Barnes CA
(1993) Thresholds for synaptic activation of transcription factors in hip-
pocampus: correlationwith long-term
Yamamoto-Sasaki M, Ozawa H, Saito T, Ro ¨sler M, Riederer P (1999) Im-
paired phosphorylation of cyclic AMP response element binding protein
in the hippocampus of dementia of the Alzheimer type. Brain Res
Zhang X, Odom DT, Koo SH, Conkright MD, Canettieri G, Best J, Chen H,
Jenner R, Herbolsheimer E, Jacobsen E, Kadam S, Ecker JR, Emerson B,
Hogenesch JB, Unterman T, Young RA, Montminy M (2005) Genome-
wide analysis of cAMP-response element binding protein occupancy,
phosphorylation, and target gene activation in human tissues. Proc Natl
Acad Sci U S A 102:4459–4464.
Zhou Y, Wu H, Li S, Chen Q, Cheng XW, Zheng J, Takemori H, Xiong ZQ
(2006) Requirement of TORC1 for late-phase long-term potentiation in
the hippocampus. PLoS ONE 1:e16.
9410 • J.Neurosci.,July14,2010 • 30(28):9402–9410Espan ˜aetal.•CRTC1SignalinginanAlzheimer’sDiseaseModel