Archival ReportBiological
Psychiatry
CRTC1 Function During Memory Encoding Is
Disrupted in Neurodegeneration
Arnaldo Parra-Damas, Meng Chen, Lilian Enriquez-Barreto, Laura Ortega, Sara Acosta,
Judith Camats Perna, M. Neus Fullana, José Aguilera, José Rodríguez-Alvarez, and
Carlos A. Saura
ABSTRACT
BACKGROUND: Associative memory impairment is an early clinical feature of dementia patients, but the molecular
and cellular mechanisms underlying these deficits are largely unknown. In this study, we investigated the functional
regulation of the cyclic adenosine monophosphate response element binding protein (CREB)–regulated transcription
coactivator 1 (CRTC1) by associative learning in physiological and neurodegenerative conditions.
METHODS: We evaluated the activation of CRTC1 in the hippocampus of control mice and mice lacking the
Alzheimer’s disease–linked presenilin genes (presenilin conditional double knockout [PS cDKO]) after one-trial
contextual fear conditioning by using biochemical, immunohistochemical, and gene expression analyses. PS cDKO
mice display classical features of neurodegeneration occurring in Alzheimer’s disease including age-dependent
cortical atrophy, neuron loss, dendritic degeneration, and memory deficits.
RESULTS: Context-associative learning, but not single context or unconditioned stimuli, induces rapid dephosphorylation (Ser151) and translocation of CRTC1 from the cytosol/dendrites to the nucleus of hippocampal neurons
in the mouse brain. Accordingly, context-associative learning induces differential CRTC1-dependent transcription of
c-fos and the nuclear receptor subfamily 4 (Nr4a) genes Nr4a1-3 in the hippocampus through a mechanism that
involves CRTC1 recruitment to CRE promoters. Deregulation of CRTC1 dephosphorylation, nuclear translocation,
and transcriptional function are associated with long-term contextual memory deficits in PS cDKO mice. Importantly,
CRTC1 gene therapy in the hippocampus ameliorates context memory and transcriptional deficits and dendritic
degeneration despite ongoing cortical degeneration in this neurodegeneration mouse model.
CONCLUSIONS: These findings reveal a critical role of CRTC1 in the hippocampus during associative memory, and
provide evidence that CRTC1 deregulation underlies memory deficits during neurodegeneration.
Keywords: Alzheimer’s disease, CREB, Gene therapy, Memory, Neurodegeneration, TORC
http://dx.doi.org/10.1016/j.biopsych.2016.06.025
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by neuropsychiatric symptoms and amnesia. Dementia
patients develop early deficits in encoding and retrieval of
associative episodic memories (1,2), a clinical feature already
present in persons at risk for developing AD (3,4). Functional
magnetic resonance imaging studies show decreased activity and connectivity of the medial temporal lobe,
particularly the hippocampus, during associative and emotional
memory tasks in AD patients (2,4–8). Memory decline in
dementia patients is accompanied by the presence of pathological features, including degeneration of synapses, dendrites,
and neurons in memory-encoding brain regions (9). Despite the
evidences of associative memory impairments and neurodegeneration in the hippocampus of dementia patients, the cellular and
molecular mechanisms linking these features are largely unclear.
Associative memories related to learning new information of
people, places, or locations are common in daily human activities.
Fear conditioning is an associative learning paradigm that allows
acquisition and consolidation of emotional-related context
memories that depends on a neural circuitry that includes the
hippocampus, amygdala, and prefrontal cortex (10). The hippocampus encodes context representations and sends projections
to the amygdala, which encodes, stores, and retrieves contextual
cues associated with aversive stimulus (11,12). Whereas different
hippocampal regions contribute to acquisition of fear contextual
memory (13,14), the CA3 subregion is activated during associative encoding and critical for initial context representations
(15,16). Besides participating in adaptive behavior, fear conditioning is implicated in the mechanisms that mediate psychopathological fear and anxiety (17), whereas dementia patients develop
associative memory impairments in fear conditioning (18,19).
The transcription factor cyclic adenosine monophosphate
response element binding protein (CREB) plays a crucial role
in contextual memory encoding, consolidation, and reconsolidation (20–22). Contextual learning induces CREB phosphorylation at Ser133 and gene transcription (23). However, CREB
phosphorylation is essential but not sufficient for gene transcription (24,25), a process that requires the specific
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ISSN: 0006-3223
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& 2016 Society of Biological Psychiatry.
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transcriptional coactivators CREB binding protein (CBP) and
CREB-regulated transcription coactivators (CRTCs). CRTCs
act as selective regulators of CREB-dependent gene expression by directing CREB occupancy to specific gene promoters
(26–28). Consistent with its role in CREB signaling, CRTC1
modulates dendritic growth, long-term synaptic plasticity, and
memory consolidation through still unclear downstream mechanisms (27,29–33). Disruption of CREB/CRTC association
impairs CREB-dependent transcription, synaptic plasticity,
and long-term memory (34), whereas CRTC1 dysfunction
causes transcriptional changes leading to memory impairments in an AD mouse model (35,36). Given this scenario, we
aimed to investigate the specific role of CRTC1 signaling in the
hippocampus during associative memory encoding in physiological and pathological conditions.
METHODS AND MATERIALS
Mice
Two- to six-month-old male mice (C57BL/6 background) and
wild-type (WT) or presenilin conditional double knockout (PS
cDKO) mice (C57BL/6/129 hybrid background) lacking expression of both PS genes (Psen1 [PS1] and Psen2 [PS2]) in
forebrain glutamatergic neurons were used (37). Littermate
control (WT; fPS1/fPS1; PS21/1 or fPS1/fPS1; PS21/–) and
PS cDKO mice (fPS1/fPS1; PS2–/–; CaMKIIα-Cre) were
obtained by crossing floxed PS1/PS2–/– (fPS1/fPS1; PS2–/–)
or PS21/– (fPS1/fPS1; PS21/–) male mice to heterozygous
PS1 cKO; PS21/– female mice (fPS1/fPS1; PS21/–; CaMKIIαCre). Experimental procedures were conducted according to
the Animal and Human Ethical Committee of the Universitat
Autònoma de Barcelona (CEEAH 1783 and 2896) following the
European Union guidelines (2010/63/EU).
Behavioral Studies
For contextual fear conditioning, mice handled for 3 days (3
min/day) were placed in a conditioning chamber (15.9 3 14 3
12.7 cm; Med Associates, St. Albans, VT) for 3 minutes, footshocked (1 s/1 mA), and retained in the chamber for 2 minutes
(immediate freezing) (38). Fear memory was tested as freezing
behavior, which was defined as a complete cessation of all
movement except for respiration, in the same conditioning
chamber for 4 minutes, 2 hours, or 24 hours after training
using Video Freeze Software (Med Associates) (Figure 1A).
Naive mice were handled but neither exposed to the conditioning chamber nor shocked, context groups were placed in
the chamber without receiving footshock, and shocked groups
were shocked and immediately returned to their home cages.
For biochemical and immunohistochemical analyses mice
were sacrificed 15 minutes after context training or memory
retention by dislocation or a lethal dose of pentobarbital,
respectively.
Adeno-Associated Virus Injections
Adeno-associated virus (AAV2/10) from rhesus macaque
(AAVrh.10) containing Crtc1-myc under the β-actin promoter
was generated by subcloning pcDNA3-Crtc1-myc (27) into
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pVAX1 (Thermo Fisher Scientific, Waltham, MA) and pGVIRES2-green fluorescent protein (GFP) vectors as described
(35). For viral injections, 4–4.5-month-old mice (n 5 6–8 mice/
group) were anesthetized with isoflurane and injected bilaterally into the dorsal hippocampus with AAV-GFP or AAVCrtc1-myc (3 mL; 5.1 3 1011 gc/mL; 0.5 mL/min). The
sterotaxic injection coordinates were as follows: anteroposterior: –2.0 mm from bregma; mediolateral: 61.8 mm from
bregma; ventral: –1.8 mm from dural surface, according to
Paxinos and Franklin mouse brain atlas. Mice were tested six
weeks after injection in contextual fear conditioning before
processed for histological and biochemical analyses.
Gene Expression Analysis
Primary neurons (4 days in vitro) were infected with scramble
or Crtc1 short hairpin RNA (ShRNA) lentiviral vectors (1–2
transducing units/cell) and treated (12 days in vitro) with
vehicle or KCl (30 mM) plus forskolin (20 μM; Sigma-Aldrich,
St. Louis, MO) for 0–12 hours. CRE luciferase assays were
performed in triplicate in at least three independent experiments (36). RNA was purified using the PureLink RNA Mini Kit
(Thermo Fisher Scientific). RNA integrity number (RIN) was
measured using the Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA). RNA (1 μg; RIN . 8.0) was
reverse-transcribed in 50 mL of a reaction mix containing 1
μM of Oligo (dT) primers, 1 μM random hexamers, 0.5 mM
dNTP, 0.45 mM DTT, RNAseOut (10 units), and SuperScript II
reverse transcriptase (Thermo Fisher Scientific) at 25ºC for 10
minutes, 42ºC for 60 minutes, and 72ºC for 10 minutes.
Quantitative real-time polymerase chain reaction (qRT-PCR)
was performed in duplicate in at least 3–5 samples using an
Applied Biosystems 7500 Fast Real-Time PCR system
(Thermo Fisher Scientific). Data analysis was performed by
the comparative ΔCt method using the Ct values and the
average value of PCR efficiencies obtained from LinRegPCR
software (http://LinRegPCR.nl) (39). Gene expression was
normalized to glyceraldehyde-3-phosphate dehydrogenase
(Gapdh) for cultured neurons or the geometric mean of Gapdh,
hypoxanthine guanine phosphoribosyl transferase (Hprt), and
peptidylprolyl isomerase A (Ppia) for brain samples (40).
Biochemical Analysis
Tissue was lysed in cold-lysis buffer (50 mM Tris hydrochloride, pH 7.4, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid, 0.5% Triton X-100, 1% NP-40, 0.1% sodium
dodecyl sulfate [SDS], 1 mM Na3VO4, 50 mM NaF, 1 mM
phenylmethylsulfonyl fluoride) containing protease and phosphatase inhibitors (Roche España, Barcelona, Spain). Proteins
were quantified with the BCA protein assay kit (Thermo Fisher
Scientific), resolved on SDS-polyacrylamide gel electrophoresis and blotted with the following antibodies: rabbit antiCRTC1 (1:5000), CREB (1:250), and phosphorylated CREB
(Ser133; 1:1000) (Cell Signaling, Danvers, MA); phosphorylated Ser151 CRTC1 (1:1000) (36) and ABE560 (Merck-Millipore, Darmstadt, Germany); and mouse anti-GAPDH (1:5000;
Abcam, Cambridge, United Kingdom). Protein bands were
quantified with ImageJ software (National Institutes of Health,
Bethesda, MD).
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Figure 1. Contextual fear learning
induces cyclic adenosine monophosphate response element binding protein (CREB)–regulated transcription
coactivator 1 (CRTC1) dephosphorylation in the hippocampus. (A) Design
of the contextual fear conditioning
(CFC) test used in this study. (B)
Freezing responses of 2–3-month-old
mice
exposed
to
context
(n 5 20) or context plus shock and
measured immediately (n 5 16), 2
hours (n 5 5), or 24 hours (n 5 5)
after training. Statistical analysis
shows a significant increase of
freezing after training (F3,42 5 9.26,
p 5 .0001). (C, D) Western blot and
quantitative analyses of CRTC1,
phosphorylated CRTC1 (pCRTC1)
(Ser151), CREB, and phosphorylated
CREB (pCREB) (Ser133) in the hippocampus of home cage (naive), context, shocked, and CFC (15 minutes, 2
hours, and 24 hours) groups. pCRTC1
levels are significantly decreased 15
minutes and 2 hours after training
(F4,16 5 4.34, p 5 .01). Values represent fold changes 6 SEM (n 5 4–5
mice/group). Statistical analysis was
determined by one-way analysis of
variance followed by Scheffé’s S (A)
or Bonferroni (B) post hoc tests.
*p , .05, **p , .01, and ***p , .0001
compared with naive mice.
Chromatin Immunoprecipitation-qPCR Analysis
Cortical neurons (12 days in vitro) were crosslinked with 1%
formaldehyde before lysis and sonication in chromatin immunoprecipitation (ChIP) buffer (50 mM Tris hydrochloride, pH
8.1, 100 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 1%
SDS, 0.1% Na deoxycholate and protease/phosphatase inhibitors). Fragmented chromatin (200–500 bp) was analyzed using
the High Sensitivity DNA Kit (Agilent Technologies). Chromatin
immunoprecipitation (2.5 μg) was performed overnight in
diluted ChIP buffer (0.1% SDS, 1.1% Triton X-100) with or
without rabbit anti-CRTC1 and CREB antibodies (Cell Signaling) (35). Input and immunoprecipitated DNA were decrosslinked and amplified by qRT-PCR using specific primers, and
the fold enrichment was calculated over an irrelevant region.
intraperitoneally) 15 minutes after contextual fear conditioning
training. Mice were perfused intracardially with 0.9% NaCl
followed by 4% buffered formalin for 2 hours. Coronal or
sagittal brain sections (5 μm) were deparaffinized in xylene,
rehydrated, and microwave heated for 10 minutes in citrate
buffer (10 mM, pH 5 6.0). Sections were incubated with rabbit
anti-CRTC1 antibody (1:300; Cell Signaling), rabbit anti-CBP
(1:200; Santa Cruz Biotechnology, Santa Cruz, CA), and
mouse NeuN (1:2000; Merck-Millipore) or microtubule associated protein 2 (MAP2) (1:200; Sigma-Aldrich) antibodies and
AlexaFluor-488/594-conjugated goat IgGs (1:400) and
Hoechst (1:10,000; Thermo Fisher Scientific). Nissl staining
was performed in floating sections (40 μm) after incubation
with cresyl violet solution (5 g/L) for 5 minutes, and cortical
thickness of somatosensory cortex was measured using
ImageJ (n 5 4–5 mice/group; n 5 3 sections/mouse).
Histological, Immunohistochemical, and
Immunofluorescence Staining
Confocal Image Acquisition and Analysis
For CRTC1 translocation analysis, mice in home conditions
or exposed to shock, context, or context plus shock were
anesthetized with a lethal dose of pentobarbital (200 mg/kg,
Images (20 3 ; zoom 0.5) obtained with a Zeiss Axio Examiner
D1 LSM700 laser scanning microscope (Carl Zeiss Microcopy,
Jena, Germany) were analyzed with ImageJ software (v.1.6x).
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CRTC1 staining intensity in the selected regions was measured using a sum projection of six Z-sections (1 μm/section).
Hoechst staining was used to determine the nuclear area,
whereas the area comprising 2 μm around the nucleus was
considered cytoplasmic CRTC1. Nuclear/cytosol CRTC1
staining intensity ratio in caudal, medial, and rostral hippocampal regions was used as measure of CRTC1 nuclear
translocation (n 5 3–4 sections/mouse; n 5 3–5 mice/group).
Dendritic CRTC1 was analyzed by quantifying CRTC1/MAP2
colocalization in the rostral CA3 hippocampus (n 5 3 sections/
mouse; n 5 8/group). Dendrite analysis was measured using
MAP2 staining intensity in a sum projection of five Z-sections
(1 μm/section) and dendritic fiber thickness was measured
automatically by generating a Plot Profile of the pixels and
peak thickness intensity along a grid line using ImageJ (n 5 3
sections/mouse; n 5 4–6 mice/group).
Statistical Analysis
Statistical analysis was performed using one- or two-way
analysis of variance (ANOVA) and Bonferroni or Tukey’s post
hoc tests for multiple comparisons using Prism software
(GraphPad, La Jolla, CA). Behavioral results were analyzed
by using two-way ANOVA with repeated measures and
Bonferroni or Scheffé’s S post hoc with SuperANOVA v1.11.
Differences with p , .05 were considered significant.
RESULTS
Contextual Fear Conditioning Induces CRTC1
Dephosphorylation, Nuclear Translocation and
Transcriptional Activity in the Hippocampus
Previous studies have shown that CRTC1 activation is mediated by activity-dependent CRTC1 dephosphorylation and
nuclear translocation (31,35,41). We first investigated the
regulation of CRTC1 by associative learning in the hippocampus, a region essential for early context representations
(15,42). Contextual fear conditioning, but not context alone,
induces a time-dependent increase of freezing responses in
mice after training, indicating efficient contextual memory
association (p , .0001, one-way ANOVA; Figure 1A, B).
Consistent with previous reports (23,43), CREB phosphorylation at Ser133 was increased in the mouse hippocampus after
context or context plus shock compared with naive conditions
(p , .05), whereas CRTC1 phosphorylation at Ser151, an
event involved in CRTC1 inactivation (33,36), was significantly
decreased 15 minutes and 2 hours after contextual training
(p , .05, one-way ANOVA; Figure 1C, D).
To explore the possibility that CRTC1 dephosphorylation
could mediate CREB-dependent transcription in the hippocampus during associative learning, we analyzed messenger
RNA (mRNA) levels of CREB target genes implicated in
contextual learning, including Arc, c-fos, and Nr4a 1, 2, and
3 (44). In agreement with previous reports, Arc is similarly
induced by a novel context and contextual conditioning
(45,46). By contrast, levels of c-fos, Nr4a1, and Nr4a2 transcripts, but not Nr4a3, are significantly increased after fear
conditioning (p , .05–.01, one-way ANOVA) but not by
context or shock alone (Figure 2A). qRT-PCR analysis revealed
that neuronal activity rapidly increases (t1/2 , 1 hours)
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transcript levels of Arc (10-fold), c-fos (40-fold), Nr4a1 (40fold), Nr4a2 (50-fold), and Nr4a3 (4-fold) (p , .001, one-way
ANOVA; Figure 2B). Because Crtc1 inactivation using Crtc1
ShRNAs significantly decreases activity-induced expression of
Arc, c-fos, Nr4a1, and Nr4a 2 (p , .05), whereas Nr4a3 mRNA
levels are not affected (Figure 2B), we explored the possibility
that CRTC1 could bind differentially to the promoter regions of
Nr4a genes. ChIP-qPCR analyses demonstrated an activitydependent recruitment of CRTC1 to the proximal CRE-TATA
promoter regions of c-fos, Nr4a1, and Nr4a2 (p , .001, oneway ANOVA) but not to the CRE-TATA-deficient region of
Nr4a3 (p . .05; Figure 2C). By contrast, CREB strongly binds
to c-fos, Nr4a1, and Nr4a2 promoters in basal nonstimulated
conditions (Figure 2C). This result suggests that activation of
CREB/CRTC1-dependent transcription is mediated by binding
of CRTC1 to proximal CRE-TATA rich gene promoters after
contextual learning in the dorsal hippocampus.
CRTC1 is mostly expressed in cell bodies and fibers of
neurons in the mouse hippocampus (CA1, CA3, and dentate
gyrus), cortex, striatum, thalamus, and amygdala (Figure 3A;
and additional data not shown). The pattern of CRTC1 staining
is similar in naive, context, or shock conditions (p . .05;
Figure 3A, C). Interestingly, CRTC1 is abundantly localized in
the nucleus of CA3 pyramidal neurons and moderately in CA1
neurons 15 minutes after contextual fear conditioning compared with in naive, context, or shock conditions (p , .01,
one-way ANOVA; Figure 3A, C and data not shown). Indeed,
CRTC1 colocalizes with MAP2 in dendrites of CA3 hippocampal neurons in naive conditions, whereas this colocalization is significantly reduced 15 minutes after contextual fear
conditioning (p , .02; Figure 3B, C). These results suggest
that contextual learning induces a rapid translocation of
CRTC1 from the cytosol and dendrites to the nucleus of
neurons in the mouse hippocampus.
Altered CRTC1-Dependent Transcription and
Nuclear Translocation Are Associated With
Contextual Memory Deficits During
Neurodegeneration
Because associative memory deficits and reduced hippocampal activity occur in AD patients (2,4–7), we next investigated
the role of CRTC1-dependent transcription in contextual fear
memory deficits in PS cDKO mice lacking both PS1 and PS2
genes in neurons of the postnatal forebrain (37). PS cDKO mice
display classical features of neurodegeneration occurring in AD,
including age-dependent cortical atrophy, enlargement of lateral ventricles, neuron loss associated with increased apoptosis
and activation of caspases 3 and 9, neuroinflammation, dendritic degeneration, and synapse loss (37,47,48). Indeed, control (WT) and PS cDKO mice at 2 months of age display similar
freezing responses 2 hours and 24 hours after contextual fear
conditioning (p . .05, two-way ANOVA; Figure 4A). By contrast,
6-month-old PS cDKO mice show reduced freezing responses
2 hours (p , .05) and 24 hours (p , .001) after contextual
training compared with control mice (two-way ANOVA;
Figure 4A), which indicates short- and long-term contextual
memory deficits in PS cDKO mice.
Gene expression analysis shows that Arc (p , .05) and
c-fos (p , .0001) transcripts are significantly increased after
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Figure 2. Contextual learning induces expression of cyclic adenosine monophosphate response element binding protein (CREB)–regulated transcription
coactivator 1 (CRTC1) target genes in the hippocampus. (A) Hippocampal messenger RNA (mRNA) levels of CREB target genes in 2–3-month-old mice in
naive, context, shock, and contextual fear conditioning (CFC) groups analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). CFC induces a
significant overall effect on hippocampal levels of Arc (F5,30 5 2.4, p 5 .05), c-fos (F5,30 5 6.7, p 5 .0003), Nr4a1 (F5,30 5 3.5, p 5 .01), and Nr4a2 (F5,30 5 2.8,
p 5 .03), but not Nr4a3 (F5,30 5 0.8, p 5 .55). Values are normalized to the geometric mean of Gapdh, Hprt1, and Ppia. Data represent mean 6 SEM (n 5 4–6
mice/group). (B) Western blot analysis of CRTC1 (top) and qRT-PCR analysis of CREB target genes normalized to Gapdh (bottom) in noninfected (NI)-,
scramble (Scr)-, and Crtc1 short hairpin RNA (ShRNA) treated hippocampal neurons. Data are the mean 6 SD of three independent experiments. (C)
Chromatin immunoprecipitation (IP) analysis of c-fos and Nr4a1, 2, and 3 gene promoters using anti-CRTC1 (left) and anti-CREB (right) antibodies in vehicle
(Veh)- and forskolin (FSK)/KCl-treated primary neurons. *p , .05, **p , .01, ***p , .001, compared with naive (A) or vehicle control (B, C), and #p , .05,
compared with FSK/KCl scramble ShRNA (B) or IP CRTC1 vehicle (C) as determined by one-way analysis of variance followed by Bonferroni (A, C) or
Dunnett’s (B) post hoc tests.
contextual training but without significant differences between
genotypes (p . .05, two-way ANOVA; Figure 4B). Nr4a1 and
Nr4a2 transcripts, but not Nr4a3, are significantly increased 24
hours after contextual learning in control and PS cDKO mice
(p , .001, two-way ANOVA) but with significant differences
between genotypes. Post hoc analysis revealed a significant
reduction of Nr4a1 (p , .001) and Nr4a2 (p , .05) transcripts,
but not those of Nr4a3, in the hippocampus of 6-month-old PS
cDKO mice at 24 hours (Figure 4B). Levels of CRTC1 are not
significantly different (p 5 .13) whereas CREB is slightly
decreased (p , .02) in the hippocampus of PS cDKO mice.
After contextual training, phosphorylated Ser151 CRTC1/
CRTC1 levels are significantly decreased in control mice
(p , .01), but not in PS cDKO mice (p 5 .47), whereas
phosphorylated CREB was increased in both WT and PS
cDKO mice, but with significant changes between genotypes
(p , .005, two-way ANOVA; Figure 4C). These results
demonstrate age-related memory impairments associated
with differential downregulation of CRTC1/CREB target genes
in the hippocampus of PS cDKO mice.
We next investigated the relationship between CRTC1
nuclear translocation and contextual memory deficits in PS
cDKO mice. Contextual fear learning induces after 15 minutes
a significant translocation of CRTC1 to the nucleus of CA3
pyramidal neurons in control (WT) mice (p , .05), whereas
CRTC1 staining is found mainly in the cytosol and sporadically
in the nucleus in PS cDKO mice (p , .05, two-way ANOVA;
Figure 5A, C). Moreover, CRTC1 is significantly decreased in
dendrites in control but not PS cDKO mice 15 minutes after
contextual learning (p , .05; two-way ANOVA; Figure 5B, C).
Together, these results suggest deficient CRTC1 nuclear
translocation and transcriptional function associated with
contextual memory deficits in PS cDKO mice.
CRTC1 Gene Therapy Ameliorates Transcriptional
and Contextual Memory Deficits in PS cDKO Mice
To evaluate whether CRTC1 dysfunction contributes to associative memory deficits in PS cDKO mice, we overexpressed
CRTC1 in vivo by using AAV2/10, a serotype that allows stable
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Figure 3. Contextual fear learning induces cyclic adenosine monophosphate response element binding protein (CREB)–regulated transcription coactivator 1
(CRTC1) dendritic delocalization and nuclear translocation in the hippocampus. (A) Confocal microscopy images showing CRTC1 (green), microtubule associated
protein 2 (MAP2) (red), and nuclear (Hoechst, blue) staining in CA3 hippocampal neurons of mice in naive or context, shocked and contextual fear conditioning (CFC)
conditions 15 minutes after training. Scale bar 5 50 μm. (B) Confocal microscopy images showing CRTC1 (green), MAP2-stained dendrites (red), and nuclei (Hoechst,
blue) in CA3 hippocampal neurons. MAP2 staining is detected as punctuate staining pattern due to its transversal position in coronal sections. Insets are magnified
images of the dashed regions showing localization (yellow) of CRTC1 in MAP2 fibers in naive conditions and its nuclear redistribution (arrowheads) 15 minutes after
CFC. Scale bar 5 50 μm. (C) Quantitative analysis of CRTC1 in the nucleus (top) and dendrites (bottom). Values represent mean 6 SEM (nucleus: n 5 4–5 mice/
group, n 5 6–12 sections/mouse; dendrites: n 5 8 mice/group, 4–6 sections/mouse). *p , .05, **p , .01, and nonsignificant (n.s.) compared with naive mice.
Statistical analysis was determined by one-way analysis of variance followed by Bonferroni post hoc test (nucleus) and t test (dendritic).
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Figure 4. Age-dependent contextual memory and cyclic adenosine monophosphate response element binding protein (CREB)–regulated transcription
coactivator 1 (CRTC1)–mediated transcription deficits in presenilin conditional double knockout (PS cDKO) mice. (A) Freezing responses of control (wild-type
[WT]) and PS cDKO mice at 2 or 6 months of age tested in contextual fear conditioning (CFC). Mice were tested immediately (context, n 5 14–24), 2 hours
(n 5 5–10), or 24 hours (n 5 5–10) after contextual training. A two-way analysis of variance (ANOVA) reveals a training effect (F3,72 5 22.6, p 5 .0001) but not a
genotype effect (F1,72 5 0.005, p 5 .94) at 2 months of age, whereas there are training (F3,121 5 25, p 5 .0001) and genotype (F1,121 5 21, p 5 .0001) effects at
6 months. (B) Hippocampal levels of messenger RNA (mRNA) in naive and CFC–trained WT and PS cDKO mice at 6 months of age. Two-way ANOVA
indicates a significant time-dependent effect for Arc (F2,29 5 6.9, p , .003), c-fos (F2,29 5 20.0, p , .0001), Nr4a1 (F2,29 5 33.0, p , .0001), and Nr4a2 (F2,29 5
27.9, p , .0001), but not Nr4a3 (F2,29 5 2.5, p 5 .1), and a genotype effect for Nr4a1 (F1,29 5 18.1, p , .0002), Nr4a2 (F1,29 5 14.8, p , .001), and Nr4a3 (F1,29
5 14.6 , p , .001), but not for Arc (F1,29 5 0.85, p 5 .36) and c-fos (F1,29 5 0.3, p 5 .6). Messenger RNA levels were quantified by quantitative real-time
polymerase chain reaction and normalized to the geometric mean of standard genes Gapdh, Hprt1, and Ppia. (C) Western blot and quantitative analyses of
CRTC1, phosphorylated CRTC1 (pCRTC1) (Ser151), CREB, and phosphorylated CREB (pCREB) (Ser133) in the hippocampus of naive and CFC (2 hours) WT
and PS cDKO mice at 6 months. Statistical analyses shows a group effect in pCREB/CREB (F1,26 5 10.1, p , .004), CREB (F1,26 5 5.4, p , .03), and pCRTC1/
CRTC1 treatment effect (F1,26 5 6.1, p , .02). Values represent mean of fold changes 6 SEM [(A, B) n 5 4–6 mice/group; (C) n 5 6–9 mice/group]. *p , .05,
**p , .005, and ***p , .0001 compared with WT mice or the indicated group. Statistical analyses were performed by two-way ANOVA followed by Scheffé’s S
(A, C) or Bonferroni (B) post hoc tests.
long-term (greater than 2 months) neuronal gene expression
(49) and enhances nuclear translocation of CRTC1-myc
and CRE-dependent transcription in cultured neurons
(Supplemental Figure S1). Four- to 4.5-month-old mice were
injected with AAV-GFP (control) and AAV-Crtc1-myc in the
CA3 hippocampus and evaluated 6 weeks later. AAV-Crtc1myc injection allowed high expression of CRTC1-myc mRNA
and protein mainly in neurons of CA1, CA3, and the dentate
gyrus (Figure 6A, B and Supplemental Figure S2). CRTC1
overexpression does not affect basal freezing responses of
WT and PS cDKO mice exposed in a novel context (p 5 .78,
two-way ANOVA; Figure 6C). By contrast, AAV-Crtc1
increases significantly freezing responses 24 hours after
contextual training both in WT (p , .05) and PS cDKO mice
(p , .03, two-way ANOVA; Figure 6D). These results suggest a
memory-enhancing effect rather than an anxiety effect of
CRTC1 overexpression in the hippocampus. Contextual fear
conditioning significantly induces Nr4a1 and Nr4a2 mRNAs in
the hippocampus of all groups (p , .001). Importantly, CRTC1
overexpression increases significantly Nr4a1 and Nr4a2
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Figure 5. Reduced translocation of
cyclic
adenosine
monophosphate
response element binding protein–regulated
transcription
coactivator
1
(CRTC1) to the nucleus of hippocampal
neurons in presenilin conditional double
knockout (PS cDKO) mice. (A) Confocal
microscopy images showing CRTC1
(green, left images) and merged
CRTC1/NeuN (red, right image) staining
in CA3 pyramidal neurons of 6-monthold wild-type (WT) and PS cDKO mice.
Arrowheads indicate some neurons
showing nuclear CRTC1. Scale bar 5
80 μm. (B) Confocal images showing
CRTC1 (green) and microtubule associated protein 2 (MAP2) (red) staining in
CA3 hippocampal neurons of 6-monthold WT and PS cDKO mice in naive and
contextual fear conditioning (CFC) (15
minutes) conditions. Arrowheads indicate nuclear CRTC1. Scale bars 5 60
μm, 15 μm (inset). (C) Quantitative analysis of nuclear (left) and dendritic (right)
CRTC1 in CA3 hippocampal neurons in
WT and PS cDKO mice. Statistical analysis shows a genotype effect on
CRTC1 nuclear localization (F1,24 5
4.03, p 5 .05). Values represent mean
6 SEM of multiple mice (n 5 4–8 mice/
group), each analyzed in multiple brain
sections (n 5 4–6/mouse). *p , .05,
compared with naive control. Statistical
analysis was determined by two-way
analysis of variance followed by Bonferroni multiple comparison post hoc test.
mRNAs in PS cDKO mice compared with GFP-injected PS
cDKO mice (p , .05, two-way ANOVA; Figure 6E). This result
indicates that CRTC1 gene therapy in the hippocampus
ameliorates transcriptional and long-term contextual memory
deficits in PS cDKO mice.
118
CRTC1 Ameliorates Dendritic Degeneration in the
Hippocampus
PS cDKO mice develop cortical neuron loss and dendritic
degeneration in the neocortex and hippocampus during
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CRTC1-Dependent Gene Expression in Neurodegeneration
Figure 6. Cyclic adenosine monophosphate response element binding
protein–regulated transcription coactivator 1 (CRTC1) gene therapy ameliorates hippocampal CRTC1-dependent
transcription and associative memory
deficits in presenilin conditional double
knockout (PS cDKO) mice. (A) Adenoassociated virus (AAV2/10)-CRTC1myc injection increases CRTC1-myc
levels in the adult mouse dorsal hippocampus. Confocal images showing
expression of exogenous green fluorescent protein (GFP) (green, left) or
CRTC1-myc (green, right) in CA3 pyramidal neurons of 6-month-old wildtype (WT) and PS cDKO mice 6 weeks
after AAV injection. Mice were sacrificed 24 hours after contextual fear
conditioning (CFC) training. Insets are
magnified images of the dashed
regions showing expression of GFP
and CRTC1-myc in NeuN-positive neurons. Hoechst (blue): nucleus. Scale bar
5 100 μm. (B) CRTC1-myc protein
(top) and messenger RNA (mRNA) (bottom) levels in the hippocampus of WT
and PS cDKO mice 6 weeks after AAV
injection. (C) Effect of CRTC1 overexpression in freezing responses in
WT and PS cDKO mice in a novel
context (n 5 6–7 mice/group). No significant differences were found among
groups (group effect: F1,21 5 1.7, p 5
.2; treatment effect: F1,21 5 0.07, p 5
.8). (D) CFC in control and PS cDKO
mice (n 5 6–7 mice/group) 6 weeks
after AAV-GFP and AAV-Crtc1 injection. Two-way ANOVA reveals significant effects of group (F3,42 5 4.3, p ,
.01), time (F1,42 5 36.8, p , .0001), and
group by time interaction (F3,42 5 3.5,
p , .02). (E) Levels of Nr4a1 and Nr4a2
transcripts in the hippocampus of AAVGFP or AAV-Crtc1 injected mice.
Values normalized to the geometric
mean of Gapdh, Hprt1, and Ppia represent mean 6 SEM (n 5 4–6 mice/
group). *p , .05, **p , .01, ***p ,
.0001, compared with WT GFP or the
indicated group. Statistical analyses
were determined by two-way analysis
of variance and Scheffé’s S (behavior)
or Bonferroni (gene expression) post
hoc tests.
aging (37,48). In agreement, 6-month-old PS cDKO mice
injected with AAV-GFP or AAV-Crtc1 in the hippocampus
show similar enlargement of lateral ventricles and reduced
cortical thickness and dendritic MAP2-stained fibers in the
neocortex (Figure 7A, B). Quantitative confocal imaging
analysis reveals significant reduction of total MAP2 staining
intensity and dendritic fibers in CA3 hippocampus of
PS cDKO-GFP mice compared with WT GFP mice (p ,
.05, two-way ANOVA; Figure 7B). Interestingly, AAV-Crtc1
increases total MAP2 intensity staining and also moderately
dendrite thickness in CA3 hippocampus of PS cDKO
mice (two-way ANOVA; Figure 7B). Notably, MAP2 staining
intensity and dendrite thickness in CA3 hippocampus
of PS cDKO-Crtc1 mice are not significantly different from
WT GFP mice (p . .05, two-way ANOVA). These results
indicate that CRTC1 gene therapy ameliorates dendritic
degeneration in the hippocampus without affecting cortical
neurodegeneration.
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Figure 7. Cyclic adenosine monophosphate response element binding protein–regulated transcription coactivator 1 (CRTC1) overexpression ameliorates
dendritic degeneration in the hippocampus of presenilin conditional double knockout (PS cDKO) mice. (A) Reduced cortical thickness in PS cDKO mice at 6
months. (Left) Nissl staining of neocortex (top) and hippocampus (bottom) showing reduced cortical thickness (dashed lines) but normal hippocampal
morphology in adeno-associated virus (AAV)-green fluorescent protein (GFP)– and AAV-Crtc1–injected PS cDKO mice. Scale bar 5 200 μm. (Right)
Quantification of cortical thickness indicates a significant group effect (F1,14 5 8.5, p , .01) but not treatment effect (F1,14 5 0.003, p 5 .96). (B) Dendritic
degeneration in the hippocampus is reduced after AAV-Crtc1 injection in PS cDKO mice. (Left) Confocal microscope images showing microtubule associated
protein 2 (MAP2)-stained fibers (red) in the neocortex (top) and CA3 hippocampus (middle) in brain sections of AAV-GFP– and AAV-Crtc1–injected mice.
Magnified dendrites in CA3 region are shown at the bottom. Scale bars: 20 μm (cortex) or 10 μm (CA3). (Right) Quantification of MAP2 staining intensity and
dendrite thickness in CA3 hippocampus. Data represent percentage of control 6 SEM of cortical thickness and MAP2 staining intensity or average of dendrite
thickness (μm) in multiple mouse brains (n 5 4–5 mice/group; n 5 3 sections/mouse). *p , .05, **p , .01, and ***p , .0001 compared with wild-type (WT) GFP
mice or the indicated group. #p 5 .05. Statistical analyses were determined by two-way analysis of variance and Scheffé’s S post hoc test.
DISCUSSION
The transcription factor CREB facilitates contextual memory
by regulating neuronal excitability and recruitment of neurons
into active memory networks (50–53). However, the CREBdependent transcriptional programs and their regulatory
mechanisms that mediate associative memory encoding are
largely unclear. In this study, we found that contextual learning
induces time-dependent dephosphorylation (Ser151), nuclear
translocation, and transcriptional activation of CRTC1 in the
hippocampus. Importantly, deregulation of CRTC1 nuclear
translocation and function in the hippocampus is associated
120
with contextual memory impairments and dendrite degeneration in a mouse model of neurodegeneration, whereas
CRTC1 gene therapy reverses these deficits. These results
strongly suggest that CRTC1-dependent transcription in the
hippocampus is critical for long-term associative memory
encoding in normal and pathological conditions.
A relevant finding of our study is that associative learning
activates CRTC1 in the hippocampus by a mechanism that
involves CRTC1 dephosphorylation and translocation from
the cytosol and dendrites to the nucleus. Contextual learning,
but not context or shock alone, induces CRTC1 nuclear
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translocation in CA3 hippocampus, and to a minor extent in
CA1 region (not shown), suggesting that CRTC1 activation in
the hippocampus can mediate rapid spatial context acquisition during memory encoding. Indeed, CRTC1 inactivation in
the hippocampus by using AAV-Crtc1 ShRNA negatively
affects long-term associative memory in control mice
(A. Parra-Damas, Ph.D., C.A. Saura, Ph.D., unpublished data,
2016). A role of CRTC1 in associative memory is further
supported by previous findings indicating that spatial memory
training induces CRTC1 nuclear translocation in the hippocampus (35), and that CRTC1 overexpression in the dorsal
hippocampus enhances contextual fear memory (30,31)
(Figure 6D). In agreement, contextual learning induces CREBmediated transcription in CA1/CA3 hippocampus, whereas
cued fear conditioning activates CREB in the amygdala (23).
Alternatively, CRTC1 is activated in the amygdala 1 day after
contextual learning (i.e., during memory consolidation) (31),
which is consistent with a role of this circuit in associating
contextual cues with aversive events (11,12). Based on these
results, we suggest that CRTC1 participates in transcriptional
events mediating contextual memory in the dorsal hippocampus, a region required for contextual memory encoding
(15,54).
Consistent with a role of CRTC1 in associative memory
encoding, contextual fear learning induces expression of
memory-related CRTC1/CREB target genes in the hippocampus. We found a time-dependent differential induction of
CREB target Nr4a family genes Nr4a1-3 in the hippocampus,
a result that agrees with the requirement of Nr4a genes for
contextual memory (44,55). CRTC1-mediated transcription
may involve CRTC1 dephosphorylation at Ser151, a critical
event for activity-induced CREB-mediated transcription
(33,36). Because the histone deacetylase CBP mediates
CREB-dependent transcription through cooperative interactions with CRTC2 and CREB (26), it is possible that CRTC1
dephosphorylation and nuclear translocation could mediate its
recruitment to CREB target promoters through cooperative
interactions with CBP and CREB. This idea is reinforced by
recent results indicating that a constitutive CRTC1 S151A/
S245A mutant enhances contextual memory by increasing
CREB-dependent transcription in the hippocampus (31). However, it is still unclear whether CBP-mediated histone acetylation plays a role on CRTC1/2/CREB complex formation.
Other alternative regulatory transcriptional mechanisms may
include kinase/phosphatase activities, synapse-nuclear translocation, acetylation, or CREB glycosylation (34,36,56,57).
Genetic and biochemical evidences suggest a role of CREB
signaling in cognitive and neurodegenerative disorders (58).
The age-related CRTC1-dependent transcription and nuclear
translocation deficits in PS cDKO mice is the first evidence
linking CRTC1 dysfunction and associative memory impairments during neurodegeneration. Although the molecular
mechanisms linking PS and CRTC1 are still largely unclear,
reduced calcium influx caused by loss of PS function (59)
could potentially lead to reduced calcineurin/PP2B activity
resulting in the observed reduced CRTC1 phosphorylation in
PS cDKO mice. Indeed, memory deficits in PS cDKO mice
were previously associated with CBP dysfunction (37), which
is consistent with fear memory deficits observed in CBPdeficient mice (60–63). Because selective expression of CREB
target genes requires cooperative interaction of CRTC/CBP
with CREB (26), a correct balance of this transcriptional
complex may be crucial for activity-dependent gene transcription during memory processing. Indeed, CBP/CRTC1/
CREB-dependent transcriptional deregulation is associated
with cognitive deficits and neurodegeneration in Huntington’s
disease (57,64). Interestingly, PS cDKO mice show contextual
memory impairments associated with hippocampal deficits of
the CRTC1 target genes Nr4a1 and Nr4a2. Particularly, Nr4a2
(Nurr1) is required for CREB-dependent neuronal survival
induced by a number of neural signals (65,66). Because Nr4a
genes (i.e., Nr4a2) are downregulated in sporadic AD and
Parkinson’s disease brains and mouse models (67), our result
may have important pathological and therapeutic implications
in neurodegenerative diseases.
Do CRTC1-dependent transcription changes contribute to
associative memory deficits in neurodegeneration? CRTC1dependent transcriptional deficits were recently associated
with early pathological and memory changes in amyloid
precursor protein transgenic mice and a rat AD model that
do not develop neurodegeneration (35,68). Pharmacological
activation of CREB signaling has been useful to reverse
memory and synaptic deficits in AD mice (69–71). Our current
gene therapy strategy indicates that enhancing specifically
CRTC1 in the hippocampus ameliorates long-term contextual
memory deficits and CRTC1-dependent transcriptional deficits
in PS cDKO mice during neurodegeneration. As shown
previously (31), CRTC1 significantly increased associative
memory although it had minor effects on Nr4a1/2 levels in
control mice. It is possible that CRTC1 overexpression in vivo
1) may affect differentially the timing of gene induction, 2)
causes a differential expression of particular set of genes as
observed after spatial memory training (35), or 3) does not
affect Nr4a1/2 levels in conditions where induction is maximal
as happens after memory training. Interestingly, CRTC1 overexpression in the hippocampus ameliorated dendrite degeneration in PS cDKO mice, suggesting a direct link between
CRTC1 dysfunction and dendrite degeneration. Although the
exact mechanism by which CRTC1 ameliorates dendrite
degeneration needs further investigation, one possibility is
that CRTC1 improves dendrite morphology through BDNF
signaling (72).
In conclusion, CRTC1 gene transfer ameliorates dendrite
degeneration, transcriptional deficits, and associative memory
symptoms during neurodegeneration. These results are highly
relevant for AD therapy because dementia patients develop
early deficits in associative memory encoding and retrieval
caused by decreased activity of the hippocampus. Targeting
CRTC1 to increase selectively expression of genes mediating
neuronal function and associative memory may represent a
promising avenue for future therapeutics in AD and other
cognitive-related disorders.
ACKNOWLEDGMENTS AND DISCLOSURES
This study was supported by grants from the Ministerio de Economia y
Competitividad of Spain (Grant Nos. SAF2013-43900-R and CIBERNED
CB06/05/0042 to CAS), Generalitat de Catalunya (Grant No. 2014 SGR0984
to JR-A) and the Alzheimer’s disease research program of the BrightFocus
Foundation (Ref. A2014417S to CAS). AP-D and MC are supported by
predoctoral fellowships from the Ministerio de Economia y Competitividad
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(Grant No. BES-2011-044405) and China Scholarship Council (CSC),
respectively.
AP-D, MC, LE-B, LO, SA, JCP, and MNF designed and performed the
experiments. AP-D, MC, LE-B, JA, JR-A and CAS contributed to data
analyses and interpreted the results. CAS coordinated the study and wrote
the paper.
We thank J. Shen (Harvard Medical School, Boston, MA) and J.-R.
Cardinaux (University of Lausanne, Swizerland) for providing the PS cDKO
mice and Crtc1-myc plasmid, respectively. We thank the technical assistance of Mar Castillo and Núria Barba from the Serveis d’Histologia i
Microscopia Units of the Institut de Neurociències, Antonio L. Florido for the
AAV injection and the Viral Vector Production Unit (UPV) and Servei de
Genòmica Bioinformàtica (SGB)-UAB.
The authors report no biomedical financial interests or potential conflicts
of interest.
12.
13.
14.
15.
16.
17.
ARTICLE INFORMATION
From the Institut de Neurociències (AP-D, MC, LE-B, LO, SA, JCP, MNF,
JA, JR-A, CAS), Department de Bioquímica i Biologia Molecular; and the
Centro de Investigación Biomédica en Red Enfermedades Neurodegenerativas (CIBERNED) (AP-D, MC, LE-B, JA, JR-A, CAS), Universitat Autònoma
de Barcelona, Bellaterra, Barcelona, Spain.
AP-D and MC contributed equally to this work.
Address correspondence to Carlos A. Saura, Ph.D., Institut de Neurociències, Facultat de Medicina M2-113, Universitat Autònoma de Barcelona, Barcelona, Spain 08193; E-mail: carlos.saura@uab.es.
Received Dec 1, 2015; revised May 31, 2016; accepted June 21, 2016.
Supplementary material cited in this article is available online at http://
dx.doi.org/10.1016/j.biopsych.2016.06.025.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
122
Granholm E, Butters N (1988): Associative encoding and retrieval in
Alzheimerʼs and Huntingtonʼs disease. Brain Cogn 7:335–347.
Sperling RA, Bates JF, Chua EF, Cocchiarella AJ, Rentz DM, Rosen
BR, et al. (2003): fMRI studies of associative encoding in young and
elderly controls and mild Alzheimerʼs disease. J Neurol Neurosurg
Psychiatry 74:44–50.
van der Meulen M, Lederrey C, Rieger SW, van Assche M, Schwartz
S, Vuilleumier P, et al. (2012): Associative and semantic memory
deficits in amnestic mild cognitive impairment as revealed by functional magnetic resonance imaging. Cogn Behav Neurol 25:195–215.
Parra MA, Pattan V, Wong D, Beaglehole A, Lonie J, Wan HI, et al.
(2013): Medial temporal lobe function during emotional memory in
early Alzheimerʼs disease, mild cognitive impairment and healthy
ageing: An fMRI study. BMC Psychiatry 13:76.
Press GA, Amaral DG, Squire LR (1989): Hippocampal abnormalities in
amnesic patients revealed by high-resolution magnetic resonance
imaging. Nature 341:54–57.
Pariente J, Cole S, Henson R, Clare L, Kennedy A, Rossor M, et al.
(2005): Alzheimerʼs patients engage an alternative network during a
memory task. Ann Neurol 58:870–879.
Small SA, Perera GM, DeLaPaz R, Mayeux R, Stern Y (1999): Differential regional dysfunction of the hippocampal formation among elderly
with memory decline and Alzheimerʼs disease. Ann Neurol 45:466–472.
Bai F, Zhang Z, Watson DR, Yu H, Shi Y, Yuan Y, et al. (2009):
Abnormal functional connectivity of hippocampus during episodic
memory retrieval processing network in amnestic mild cognitive
impairment. Biol Psychiatry 65:951–958.
Spires-Jones TL, Hyman BT (2014): The intersection of amyloid beta
and tau at synapses in Alzheimerʼs disease. Neuron 82:756–771.
Maren S, Phan KL, Liberzon I (2013): The contextual brain: Implications for fear conditioning, extinction and psychopathology. Nat Rev
Neurosci 14:417–428.
Sanders MJ, Wiltgen BJ, Fanselow MS (2003): The place of the
hippocampus in fear conditioning. Eur J Pharmacol 463:217–223.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
Maren S (2008): Pavlovian fear conditioning as a behavioral assay for
hippocampus and amygdala function: Cautions and caveats. Eur J
Neurosci 28:1661–1666.
Small SA, Nava AS, Perera GM, DeLaPaz R, Mayeux R, Stern Y
(2001): Circuit mechanisms underlying memory encoding and retrieval
in the long axis of the hippocampal formation. Nat Neurosci 4:
442–449.
Zeineh MM, Engel SA, Thompson PM, Bookheimer SY (2003):
Dynamics of the hippocampus during encoding and retrieval of
face-name pairs. Science 299:577–580.
Lee I, Kesner RP (2004): Differential contributions of dorsal hippocampal subregions to memory acquisition and retrieval in contextual
fear-conditioning. Hippocampus 14:301–310.
Phillips RG, LeDoux JE (1995): Lesions of the fornix but not the
entorhinal or perirhinal cortex interfere with contextual fear conditioning. J Neurosci 15:5308–5315.
Bishop S (2007): Neurocognitive mechanisms of anxiety: An integrative account. Trends Cogn Sci 11:307–316.
Hamann S, Monarch E, Golstein F (2002): Impaired fear conditioning in
Alzheimerʼs disease. Neuropsychologia 40:1187–1195.
Hoefer M, Allison S, Schauer G, Neuhaus J, Hall J, Dang J, et al.
(2008): Fear conditioning in frontotemporal lobar degeneration and
Alzheimerʼs disease. Brain 131:1646–1657.
Frankland PW, Josselyn SA, Anagnostaras SG, Kogan JH, Takahashi
E, Silva AJ (2004): Consolidation of CS and US representations in
associative fear conditioning. Hippocampus 14:557–569.
Kida S, Josselyn SA, Pena de Ortiz S, Kogan JH, Chevere I,
Masushige S, et al. (2002): CREB required for the stability of new
and reactivated fear memories. Nat Neurosci 5:348–355.
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.
Impey S, Smith DM, Obrietan K, Donahue R, Wade C, Storm DR
(1998): Stimulation of cAMP response element (CRE)-mediated transcription during contextual learning. Nat Neurosci 1:595–601.
Chrivia JC, Kwok RPS, Lamb N, Hagiwara M, Montminy MR, Goodman RH (1993): Phosphorylated CREB binds specifically to the
nuclear protein CBP. Nature 365:855–859.
Bito H, Deisseroth K, Tsien RW (1996): CREB phosphorylation and
dephosphorylation: a Ca21- and stimulus duration-dependent switch
for hippocampal gene expression. Cell 87:1203–1214.
Ravnskjaer K, Kester H, Liu Y, Zhang X, Lee D, Yates III JR, et al.
(2007): Cooperative interactions between CBP and TORC2 confer
selectivity to CREB target gene expression. EMBO J 26:2880–2889.
Kovács KA, Steullet P, Steinmann M, Do KQ, Magistretti PJ, Halfon O,
et al. (2007): TORC1 is a calcium- and cAMP-sensitive coincidence
detector involved in hippocampal long-term synaptic plasticity. Proc
Natl Acad Sci U S A 104:4700–4705.
Conkright MD, Canettieri G, Screaton R, Guzman E, Miraglia L,
Hogenesch JB, et al. (2003): TORCs: Transducers of regulated CREB
activity. Moll Cell 12:413–423.
Hirano Y, Masuda T, Naganos S, Matsuno M, Ueno K, Miyashita T,
et al. (2013): Fasting launches CRTC to facilitate long-term memory
formation in Drosophila. Science 339:443–446.
Sekeres MJ, Mercaldo V, Richards B, Sargin D, Mahadevan V, Woodin
MA, et al. (2012): Increasing CRTC1 function in the dentate gyrus
during memory formation or reactivation increases memory strength
without compromising memory quality. J Neurosci 32:17857–17868.
Nonaka M, Kim R, Fukushima H, Sasaki K, Suzuki K, Okamura M,
et al. (2014): Region-specific activation of CRTC1-CREB signaling
mediates long-term fear memory. Neuron 84:92–106.
Li S, Zhang C, Takemori H, Zhou Y, Xiong ZQ (2009): TORC1
regulates activity-dependent CREB-target gene transcription and
dendritic growth of developing cortical neurons. J Neurosci 29:
2334–2343.
Altarejos JY, Goebel N, Conkright MD, Inoue H, Xie J, Arias CM, et al.
(2008): The Creb1 coactivator Crtc1 is required for energy balance and
fertility. Nat Med 14:1112–1117.
Biological Psychiatry January 15, 2017; 81:111–123 www.sobp.org/journal
Biological
Psychiatry
CRTC1-Dependent Gene Expression in Neurodegeneration
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
Rexach JE, Clark PM, Mason DE, Neve RL, Peters EC, Hsieh-Wilson
LC (2012): Dynamic O-GlcNAc modification regulates CREB-mediated
gene expression and memory formation. Nat Chem Biol 8:253–261.
Parra-Damas A, Valero J, Meng C, España J, Martin E, Ferrer I, et al.
(2014): Crtc1 activates a transcriptional program deregulated at early
Alzheimer’s disease-related stages. J Neurosci 34:5776–5787.
España J, Valero J, Miñano-Molina AJ, Masgrau R, Martín E, GuardiaLaguarta C, et al. (2010): β-Amyloid disrupts activity-dependent gene
transcription required for memory through the CREB coactivator
CRTC1. J Neurosci 30:9402–9410.
Saura CA, Choi SY, Beglopoulos V, Malkani S, Zhang D, Shankaranarayana Rao BS, et al. (2004): Loss of presenilin function causes
impairments of memory and synaptic plasticity followed by agedependent neurodegeneration. Neuron 42:23–36.
España J, Gimenez-Llort L, Valero J, Miñano A, Rabano A, RodriguezAlvarez J, et al. (2010): Intraneuronal β-amyloid accumulation in the
amygdala enhances fear and anxiety in Alzheimerʼs disease transgenic mice. Biol Psychiatry 67:513–521.
Ruijter JM, Ramakers C, Hoogaars WM, Karlen Y, Bakker O, van den
Hoff MJ, et al. (2009): Amplification efficiency: Linking baseline and bias
in the analysis of quantitative PCR data. Nucleic Acids Res 37:e45.
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M,
et al. (2009): The MIQE guidelines: Minimum information for publication
of quantitative real-time PCR experiments. Clin Chem 55:611–622.
Chʼng TH, DeSalvo M, Lin P, Vashisht A, Wohlschlegel JA, Martin KC
(2015): Cell biological mechanisms of activity-dependent synapse to
nucleus translocation of CRTC1 in neurons. Front Mol Neurosci 8:48.
Phillips RG, LeDoux JE (1992): Differential contribution of amygdala
and hippocampus to cued and contextual fear conditioning. Behavioral Neuroscience 106:274–285.
Stanciu M, Radulovic J, Spiess J (2001): Phosphorylated cAMP
response element binding protein in the mouse brain after fear
conditioning: Relationship to Fos production. Brain Res Mol Brain
Res 94:15–24.
Hawk JD, Bookout AL, Poplawski SG, Bridi M, Rao AJ, Sulewski ME,
et al. (2012): NR4A nuclear receptors support memory enhancement
by histone deacetylase inhibitors. J Clin Invest 122:3593–3602.
Huff NC, Frank M, Wright-Hardesty K, Sprunger D, Matus-Amat P,
Higgins E, et al. (2006): Amygdala regulation of immediate-early gene
expression in the hippocampus induced by contextual fear conditioning. J Neurosci 26:1616–1623.
Pevzner A, Miyashita T, Schiffman AJ, Guzowski JF (2012): Temporal
dynamics of Arc gene induction in hippocampus: Relationship to
context memory formation. Neurobiol Learn Mem 97:313–320.
Watanabe H, Iqbal M, Zheng J, Wines-Samuelson M, Shen J (2014):
Partial loss of presenilin impairs age-dependent neuronal survival in
the cerebral cortex. J Neurosci 34:15912–15922.
Wines-Samuelson M, Schulte EC, Smith MJ, Aoki C, Liu X, Kelleher
RJ 3rd, et al. (2010): Characterization of age-dependent and progressive cortical neuronal degeneration in presenilin conditional
mutant mice. PLoS One 5:e10195.
Klein RL, Dayton RD, Tatom JB, Henderson KM, Henning PP (2008):
AAV8, 9, Rh10, Rh43 vector gene transfer in the rat brain: Effects of
serotype, promoter and purification method. Mol Ther 16:89–96.
Viosca J, Lopez de Armentia M, Jancic D, Barco A (2009): Enhanced
CREB-dependent gene expression increases the excitability of neurons in the basal amygdala and primes the consolidation of contextual
and cued fear memory. Learn Mem 16:193–197.
Suzuki A, Fukushima H, Mukawa T, Toyoda H, Wu LJ, Zhao MG, et al.
(2011): Upregulation of CREB-mediated transcription enhances both
short- and long-term memory. J Neurosci 31:8786–8802.
Restivo L, Tafi E, Ammassari-Teule M, Marie H (2009): Viral-mediated
expression of a constitutively active form of CREB in hippocampal
neurons increases memory. Hippocampus 19:228–234.
Won J, Silva AJ (2008): Molecular and cellular mechanisms of memory
allocation in neuronetworks. Neurobiol Learn Mem 89:285–292.
Ramamoorthi K, Fropf R, Belfort GM, Fitzmaurice HL, McKinney RM,
Neve RL, et al. (2011): Npas4 regulates a transcriptional program in
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
CA3 required for contextual memory formation. Science 334:
1669–1675.
Rojas P, Joodmardi E, Hong Y, Perlmann T, Ogren SO (2007): Adult
mice with reduced Nurr1 expression: An animal model for schizophrenia. Mol Psychiatry 12:756–766.
Chʼng TH, Uzgil B, Lin P, Avliyakulov NK, OʼDell TJ, Martin KC (2012):
Activity-dependent transport of the transcriptional coactivator CRTC1
from synapse to nucleus. Cell 150:207–221.
Jeong H, Cohen DE, Cui L, Supinski A, Savas JN, Mazzulli JR, et al.
(2012): Sirt1 mediates neuroprotection from mutant huntingtin by
activation of the TORC1 and CREB transcriptional pathway. Nat Med
18:159–165.
Saura CA, Valero J (2011): The role of CREB signaling in
Alzheimer’s disease and other cognitive disorders. Rev Neurosci
22:153–169.
Zhang C, Wu B, Beglopoulos V, Wines-Samuelson M, Zhang D,
Dragatsis I, et al. (2009): Presenilins are essential for regulating
neurotransmitter release. Nature 460:632–636.
Alarcon JM, Malleret G, Touzani K, Vronskaya S, Ishii S, Kandel ER,
et al. (2004): Chromatin acetylation, memory, and LTP are impaired in
CBP(1/-) mice: A model for the cognitive deficit in Rubinstein-Taybi
syndrome and its amelioration. Neuron 42:947–959.
Bourtchouladze R, Lidge R, Catapano R, Stanley J, Gossweiler S,
Romashko D, et al. (2003): A mouse model of RubinsteinTaybi syndrome: Defective long-term memory is ameliorated by
inhibitors of phosphodiesterase 4. Proc Natl Acad Sci U S A 100:
10518–10522.
Barrett RM, Malvaez M, Kramar E, Matheos DP, Arrizon A, Cabrera
SM, et al. (2011): Hippocampal focal knockout of CBP affects specific
histone modifications, long-term potentiation, and long-term memory.
Neuropsychopharmacology 36:1545–1556.
Chen G, Zou X, Watanabe H, van Deursen JM, Shen J (2010): CREB
binding protein is required for both short-term and long-term memory
formation. J Neurosci 30:13066–13077.
Giralt A, Puigdellivol M, Carreton O, Paoletti P, Valero J, Parra-Damas
A, et al. (2012): Long-term memory deficits in Huntingtonʼs disease
are associated with reduced CBP histone acetylase activity. Hum Mol
Genet 21:1203–1216.
Barneda-Zahonero B, Servitja JM, Badiola N, Minano-Molina AJ, Fado
R, Saura CA, et al. (2012): Nurr1 protein is required for N-methyl-Daspartic acid (NMDA) receptor-mediated neuronal survival. J Biol
Chem 287:11351–11362.
Volakakis N, Kadkhodaei B, Joodmardi E, Wallis K, Panman L,
Silvaggi J, et al. (2010): NR4A orphan nuclear receptors as mediators
of CREB-dependent neuroprotection. Proc Natl Acad Sci U S A 107:
12317–12322.
Skerrett R, Malm T, Landreth G (2014): Nuclear receptors in neurodegenerative diseases. Neurobiol Dis 72 Pt A:104–116.
Wilson EN, Abela AR, Do Carmo S, Allard S, Marks AR, Welikovitch
LA, et al. (2016): Intraneuronal amyloid beta accumulation disrupts
hippocampal CRTC1-dependent gene expression and cognitive function in a rat model of Alzheimer disease [published online ahead of
print Jan 11]. Cereb Cortex.
Yiu AP, Rashid AJ, Josselyn SA (2011): Increasing CREB function in
the CA1 region of dorsal hippocampus rescues the spatial memory
deficits in a mouse model of Alzheimerʼs disease. Neuropsychopharmacology 36:2169–2186.
Gong B, Vitolo OV, Trinchese F, Liu S, Shelanski M, Arancio O (2004):
Persistent improvement in synaptic and cognitive functions in an
Alzheimer mouse model after rolipram treatment. J Clin Invest 114:
1624–1634.
Caccamo A, Maldonado MA, Bokov AF, Majumder S, Oddo S (2011):
CBP gene transfer increases BDNF levels and ameliorates learning
and memory deficits in a mouse model of Alzheimerʼs disease. Proc
Natl Acad Sci U S A 107:22687–22692.
Finsterwald C, Fiumelli H, Cardinaux JR, Martin JL (2010): Regulation
of dendritic development by BDNF requires activation of CRTC1 by
glutamate. J Biol Chem 285:28587–28595.
Biological Psychiatry January 15, 2017; 81:111–123 www.sobp.org/journal
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BSCI343: CELLULAR MECHANISMS OF AGING & DISEASE
SPRING 2022
Alzheimer’s Disease Research Article Discussion Questions (due by 11:59 pm 5/2/22)
1. In your own words, what was the specific purpose of this study (what did the authors seek to
determine and why did they want to do this)?
2. Please describe, in your own words, how contextual fear conditioning was performed on the
mice in this study. Include how ‘fear’ was actually measured, and distinguish among the other
control groups used (naïve groups, context groups, and shocked groups).
3. What are CREB and CRTC1, and how specifically are they thought to be relevant to learning and
memory?
4. According to the study, how is CREB’s activity regulated by its phosphorylation status? Contrast
this with CRTC1’s activity.
5.
Provide an overview and explain the significance of the authors’ findings in Figures 1B-1D.
6. Use an outside source (provide the URLs for the sources you use for this) to learn the basic
function of the genes below. The explain why the authors wanted to measure gene expression
of these genes, as well as what they found overall (Figure 2).
a. Arc
b. C-fos
c. Nrf4a gene family
7. Why were the authors interested in measuring CRCT1/CREB activity in Alzheimer’s disease, and
what specific type of mice did they use to model this?
8. In Figure 3, what happened to CRTC1’s localization in fear conditioned vs context vs shocked
mice? Why was this significant, and how did this compare to the findings in Figure 5?
9. Please provide a detailed explanation of the authors’ findings from any three subfigures in
Figure 6. Be sure to include what was being measured.
10. What are 3 conclusions that came from this study? What are two hypothetical experiments that
would you like to see done next, in order to determine whether this study’s findings could be
potentially helpful in diagnosing or treating Alzheimer’s in humans?
11. Finally, this article is entitled, CRTC1 Function During Memory Encoding is Disrupted n
Neurodegeneration, but please provide your own alternative title to this article, based upon
your understanding of the article’s findings
.
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