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Published as doi: 10.1096/fj.08-107888.
 (The FASEB Journal. 2008;22:2872-2879.)
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CREB has a context-dependent role in activity-regulated transcription and maintains neuronal cholesterol homeostasis

Thomas Lemberger*,1,2, Jan Rodriguez Parkitna*,1, Minqiang Chai*, Günther Schütz*,3 and David Engblom*,{dagger}

* German Cancer Research Center, Heidelberg, Germany; and

{dagger} Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden

3Correspondence: German Cancer Research Center, 69120 Heidelberg, Germany. E-mail: g.schuetz{at}dkfz.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Induction of specific gene expression patterns in response to activity confers functional plasticity to neurons. A principal role in the regulation of these processes has been ascribed to the cAMP responsive element binding protein (CREB). Using genome-wide expression profiling in mice lacking CREB in the forebrain, accompanied by deletion of the cAMP responsive element modulator gene (CREM), we here show that the role of these proteins in activity-induced gene expression is surprisingly selective and highly context dependent. Thus, only a very restricted subset of activity-induced genes (i.e., Gadd45b or Nr4a2) requires these proteins for their induction in the hippocampus after kainic acid administration, while they are required for most of the cocaine-induced expression changes in the striatum. Interestingly, in the absence of CREB, CREM is able to rescue activity-regulated transcription, which strengthens the notion of overlapping functions of the two proteins. In addition, we show that cholesterol metabolism is dysregulated in the brains of mutant mice, as reflected coordinated expression changes in genes involved in cholesterol synthesis and neuronal accumulation of cholesterol. These findings provide novel insights into the role of CREB and CREM in stimulus-dependent transcription and neuronal homeostasis.—Lemberger, T., Parkitna, J. R., Chai, M., Schütz, G., Engblom, D. CREB has a context-dependent role in activity-regulated transcription and maintains neuronal cholesterol homeostasis


Key Words: cocaine • plasticity • microarray • Cre/loxP • neurodegeneration


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
STRONG AND PATTERNED STIMULATION of neurons triggers changes in synaptic plasticity and increases the expression of several transcripts, such as Fos or Egr1 (1 2 3) . Importantly, plasticity is highly dependent on early changes in gene expression (4) . Thus, stabilization of the late phase of hippocampal LTP is suppressed when gene expression is blocked (5) . By extension, perturbation of protein or RNA synthesis and deletion of specific activity-regulated genes interfere with the formation of long-term memory (4 , 6) .

Collectively, these findings have prompted intense research to identify transcriptional pathways that mediate the genomic effect of neuronal activity (7) . This is a complex task because many parallel transcription pathways converge onto the promoters of activity-dependent genes. This is well illustrated by the promoter of the Fos gene, which harbors binding sites for the cAMP-response element binding protein (CREB), the serum response factor (SRF), ELK1, and the signal transducer and activator of transcription (STAT) family of proteins (8) .

CREB was one of the first stimulus-dependent transcription factors to be cloned (9) , and it has been suggested to play a particularly important role in neuronal and behavioral plasticity (10 11 12) . The contribution of CREB in activity-dependent transcription has been intensively studied in vitro (13 14 15) . Further, classical promoter analysis and genome-wide location studies have identified CREB-binding sites in the promoters of thousands of genes, including many activity-dependent genes (12 , 16 17 18) . However, as these promoters also harbor binding sites for many other stimulus-dependent transcription factors expressed in neurons, it is unclear to what extent CREB signaling contributes to activity-induced transcription in the living brain.

CREB and the cAMP responsive element modulator (CREM; a transcription factor in the same family as CREB that compensates for CREB deficiency) are also important for neuronal survival (19 , 20) . Thus, mice lacking CREB and CREM in the forebrain show neurodegeneration, primarily in the hippocampus and striatum (21) . However, the gene expression changes that precede, and potentially cause, the neurodegenerative process remain to be elucidated.

Previous animal studies on CREB/CREM-dependent transcription in the brain have been limited to the use of dominant-negative CREB variants, which reduce but do not abolish the activity of these proteins (10 , 22) . In the present study, we examined how loss of CREB and CREM affects gene expression in the brain, using genome-wide expression profiling in two different models of neuronal activation. We show that CREB/CREM signaling has a highly context dependent and surprisingly specific role in activity-regulated gene expression. Intriguingly, we also found that loss of CREB/CREM leads to deregulated cholesterol metabolism and a pathological accumulation of cholesterol.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal experiments
Animal experiments were carried out on transgenic animals with targeted mutation of the Creb1 gene in which the exons carrying the DNA binding domain are flanked with loxP sites crossed with the transgenic line carrying the Cre recombinase expressed under the control of the Camk2a promoter (21) and a deletion of the Crem gene (23) . The Creb1Camkcre4; Crem–/– ("mutant"), Creb1Camkcre4; Crem+/–, and Creb1loxP/loxP; Crem+/– ("control") animals were bred in a mixed C57BL6/N and 129 background. All animal procedures were approved by the German animal welfare office of the Regierungspräsidium Karlsruhe, Germany.

For the characterization of CREB/CREM-dependent genes, 5- to 6-wk-old littermate male and female mice were single-caged, handled for 5 days, and then injected i.p. with either saline, kainic acid (20 mg/kg), or cocaine (25 mg/kg; Sigma, St. Louis, MO, USA). One hour after injection, animals were killed by cervical dislocation, and the hippocampus or whole brain was removed and placed in RNAlater (Ambion, Austin, TX, USA) overnight. Brains were sliced into 150-µm-thick vibratome sections, and the striatum including the nucleus accumbens was excised with thin needles. Total RNA was prepared with either the Absolutely RNA Miniprep kit (Stratagene, La Jolla, CA, USA) or Rneasy RNA Mini kit (Qiagen, Valencia, CA, USA) and its quality was assessed on RNA LabChips (Agilent, Santa Clara, CA, USA).

The kainic acid injections were replicated independently 3 times and conducted each time on 3 to 5 Creb1Camkcre4; Crem–/– animals and 3 to 5 Creb1loxP/loxP; Crem+/– littermates for both saline and drug treatments. Successful induction was verified on each sample by real-time quantitative polymerase chain reaction (PCR) measurement of Ier2, an immediate-early gene found to be CREB/CREM independent (data not shown). RNA samples within each group were pooled, resulting in 3 saline-treated control/mutant pairs and 3 kainic acid-treated control/mutant pairs. In the case of the cocaine-dependent induction of gene expression in the striatum, samples were not pooled. Groups of 3 control and mutant mice were injected with saline, and cocaine injections were given to groups of 5 control and mutant mice, as well as to 3 Creb1Camkcre4; Crem+/– animals.

DNA microarray expression profiling
Microarray experiments were carried out using GeneChip MG-U74Av2, MG-U74Bv2, and MG-U74Cv2 arrays (Affymetrix, Santa Clara, CA, USA) for the hippocampus and 430A 2.0 arrays for the striatum. All procedures were performed according to the manufacturer’s instruction. A total of 36 arrays were hybridized with RNA from the hippocampus (kainic acid and saline treatments), and 19 arrays were hybridized with samples from the striatum (cocaine and saline treatments). Analysis of array data was performed using the R 2.4.1/Bioconductor 1.9 (24) . Data was normalized and expression values were computed using the gcrma method. Statistical analysis was performed by empirical Bayes inference for linear models using the limma package. Gene set enrichment analysis was performed with the GSEAv2 package (25) (http://www.broad.mit.edu/gsea/) and Gene Ontology (GO) analysis with the BINGO plugin to the Cytoscape software (http://www.cytoscape.org) with inclusion criteria fold-change > 1.5 and P < 0.001 for the striatal dataset and P < 0.05 for the hippocampal dataset (different criteria were used in order to include reasonably similar number of genes in the analysis). All raw array data were deposited with the Gene Expression Omnibus database (U.S. National Center for Biotechnology Information, Bethesda, MD, USA) under accession numbers GSE8944, GSE8946, GSE8947, and GSE8948.

RNA interference
ST14A cells (26) were transfected with 25 nM of control nonsilencing siRNA [Qiagen; sense: r(UUCUCCGAACGUGUCACGU)d(TT)] or 12.5 nM of Creb1-silencing [sense: r(GCAAGAGAAUGUCGUAGAA)d(TT)] and 12.5 nM of Crem-silencing siRNA [sense: r(CAGGUGACAUGCCAACUUA)d(TT)] using TransIT-TKO transfection reagent (Mirus, Madison, WI, USA). On the second day after transfection, cells were shifted to the nonpermissive temperature (39°C). On the third day, cells were induced by either vehicle (dimethyl sulfoxide), 200 nM phorbol 12-myristate 13-acetate (PMA; Sigma) or 50 mM forskolin (Sigma) for 1 h. RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA), reverse transcribed (First strand synthesis kit, Invitrogen) and analyzed by real-time PCR using TaqMan gene expression assays (Applied Biosciences, Foster City, CA, USA).

Cholesterol staining
Mutant and control animals were killed and perfused with 4% (w/v) paraformaldehyde. Brain sections (50 µm) were washed and stained with 10 µg/ml Filipin III (Sigma) in PBS for 4 h at room temperature. Subsequently, the sections were washed and mounted in aqueous medium. Fluorescence images were acquired with UV lamp illumination.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The role of CREB and CREM in activity-regulated transcription depends on the context
We used two models of activity regulated-transcription: cocaine-induced transcription in the striatum and kainic acid-induced transcription in the hippocampus. Both treatments have been shown to produce strong and long-lasting plasticity changes and induction of gene expression (17) . One hour after cocaine administration, 42 different transcripts were induced in the striatum to a significant extent (P<0.001; fold change>1.5) (Fig. 1 A). In response to kainic acid, 57 transcripts were significantly induced (P<0.001; and fold change>1.5) in the hippocampus after 1 h (Fig. 1B ). Although the profiles of gene induction were not identical between the two treatments they overlapped to a significant extent, including most of the known immediate-early genes (Supplemental Tables S1 and S2).


Figure 1
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  		Figure 1. Role of CREB in activity-regulated gene expression. Heat maps represent a summary of activity-induced transcripts (fold-change>1.5, P<0.001) in the striatum 1 h after i.p. injection with 25 mg/kg cocaine (A) or in the hippocampus, 1 h after i.p. treatment with 20 mg/kg kainic acid (B). Each row corresponds to the transcript of the gene indicated on the right. The "ht" and "mutant" labels indicate results from the analysis of Creb1Camkcre4; Crem+/– and Creb1Camkcre4; Crem–/–, respectively. Gene names in bold correspond to transcripts significantly induced by treatment in both the hippocampus and the striatum of controls. Colors are proportional to the log2 of the normalized array result, as indicated on the scale below the panel. Transcripts are ordered by t-statistic from comparison of induced mutants vs. controls, with the most affected by CREB and CREM loss at top and least affected at bottom. Transcripts with different abundance (P<0.05) in induced controls compared to induced mutants are in the dotted-line boxes as indicated by labels. See Supplemental Tables S1 and S2 for detailed lists.

To evaluate the contribution of CREB to neuronal activity-dependent gene expression, we analyzed the activity-regulated gene transcription in the Creb1Camkcre4; Crem–/– transgenic mice. In these animals, the deletion of exons 9 and 10 of the Creb1 gene using the Cre/loxP mechanism occurs postnatally in forebrain neurons (21) . The Crem–/– background is necessary to prevent compensation of Creb1 loss by Crem overexpression (27) . We used mice at the age of 5–6 wk in order to minimize the extent of neurodegeneration, which at this age is minimal (21) . Loss of Crem alone has minimal effect on activity-regulated transcription (data not shown).

In the striatum, the double ablation of Creb1 and Crem produced an almost complete loss of activity-regulated transcription (Fig. 1A ; Supplemental Table S1), consistently with the role attributed to CREB. Of the 42 mRNAs induced significantly in control animals, only two transcripts, Egr2 and Nr4a1, were still significantly increased in the double mutants after cocaine treatment. All the remaining activity-regulated genes failed to be induced or were only slightly increased (Fig. 1A ; Supplemental Table S1). Furthermore the presence of a single Crem allele in the Creb1Camkcre4; Crem+/– animals was sufficient to substantially rescue the cocaine-induced transcription in the striatum (Fig. 1 ; Supplemental Table S1). For instance, the abundance of the Fos transcript was increased 8.9-fold, as compared to 16-fold in control animals and 1.4-fold in Creb1Camkcre4; Crem–/– mice. A similar trend was observed in cases of Junb or Tiparp. Nevertheless, the presence of a Crem allele did not restore the induction of all activity-regulated genes. The abundances of transcripts such as Siah2, Gadd45b, or Gpr19 were not significantly changed from basal levels after cocaine injection in the Creb1Camkcre4; Crem+/– mice.

Interestingly, kainic acid-induced gene expression in the hippocampus was largely unaffected by the combined ablation of Creb1 and Crem (Fig. 1B ; Supplemental Table S2). Of the 57 different transcripts significantly induced in the controls, 30 transcripts, including Fosb, Fos, Egr1, and Egr2, were still induced to the similar level in the mutant mice. Several transcripts showed mildly reduced induction in the mutant mice, whereas very few genes displayed significantly attenuated induction in the mutants, most notably Gadd45b, Gpr19, Nfil19, Nr4a2, Atf3, and Spty2d1. Similar results were obtained from the analysis of microdissected CA3 region (data not shown), which does not undergo neurodegeneration in the Creb1Camkcre4; Crem–/– mice (21) . A validation of both striatum and hippocampus array data performed for selected transcripts using quantitative PCR indicated good reproducibility (Supplemental Fig. S1). These results show that the role of CREB in activity-dependent transcription is highly dependent on the stimulus used and the brain region investigated and reveals that, surprisingly, CREB/CREM function is almost dispensable in the context of kainic acid-induced hippocampal gene expression.

Dependency on CREB signaling is cell autonomous
In the in vivo experiments above, both the nature of the stimulus and the targeted brain region were varied. To examine whether the context dependency of CREB function can be observed within a single cell population and cell autonomously, we performed simultaneous knockdown of Creb1 and Crem in the immortalized striatal ST14A cell line (26) (Fig. 2 A), since loss of CREB results in a compensatory two-fold up-regulation of Crem transcript. One hour after the ST14A cells were treated with the adenylyl cyclase activator forskolin, the expression of Nr4a2 and Fos was robustly induced, whereas no significant change in Egr2 transcript was observed (Fig. 2B ). Induction by the protein kinase C activator PMA strongly stimulated expression of Fos and Egr2 but not Nr4a2. Similar to the situation in the living striatum, down-regulation of Creb1 and Crem interfered with forskolin-induction of the three immediate-early genes, showing that the effects observed in vivo in the striatum can be reproduced in vitro and are cell autonomous. Interestingly, Fos and Egr2 induction by PMA was unaffected by reduction in CREB and CREM levels, illustrating that also within a single cell type, the involvement of the CREB signaling pathway is highly dependent on the stimulus chosen.


Figure 2
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  		Figure 2. In vitro silencing of Creb1 and Crem. A) Silencing of Creb1 (siCreb1) in ST14A cells causes concomitant up-regulation of Crem expression. Simultaneous RNAi against Creb1 and Crem (siCrem) prevents the compensatory effects of Crem. B) Quantitative real-time PCR measurements of expression levels of Nr4a2, Fos, and Egr2 in ST14A cells 1 h after treatment with vehicle (ctrl), 200nM PMA, or 50 µM forskolin (F). Cells were transfected either with nonsilencing siRNA (white bars) or with Creb1 and Crem-silencing siRNA (black bars). Average fold induction (n=3) relative to vehicle-treated nonsilencing siRNA-transfected cells is indicated. Silencing of Creb1 and Crem impairs the forskolin-triggered induction of Nr4a2 and Fos.

Impaired cholesterol homeostasis in CREB/CREM-deficient neurons
Our analysis so far has concentrated on activity-dependent genes. However, genome-wide expression profiling provides the opportunity to more broadly explore the biological processes that are affected in the brains of CREB/CREM mutants in an unbiased way. To detect coordinated expression changes of genes belonging to specific pathways or functional categories, we tested for overrepresentation of GO functional terms ("biological process") and also analyzed our datasets using the Gene Set Enrichment Analysis (GSEA) method. This analysis suggests that many genes associated with immune defense, inflammation, and cell death are up-regulated in CREB/CREM mutant animals as compared to their respective controls (Table 1 ; Supplemental Tables S3 to S12). These observations are consistent with the onset of the neurodegenerative process in mutant animals at this age and with the resulting reactive astrogliosis. Indeed, genes including Gfap, Spp1, and Lyzs, all known to be associated with astrocyte and microglia activation (28) , are among the genes that are the most strongly affected by the loss of CREB and CREM (Supplemental Tables S3 and S4). The outcome of the analysis of gene sets down-regulated in CREB/CREM-deficient brains was more surprising. Indeed, one of the top-ranking pathways identified by both the GO term enrichment analyses and GSEA turned out to be the cholesterol biosynthesis pathway (Table 1 ; Supplemental Tables S6, S8, S10, and S12). In particular, the expression levels of Cyp51, Hmgcs, Hmgcr, Idi1, and Sc4mol were collectively attenuated (Fig. 3 A; Supplemental Table S13). Intriguingly, several genes involved in cholesterol trafficking or efflux, such as Npc2, ApoE, Cyp46a1, Ch25h, and Abca1, were also changed (P<0.001) in mutant brains (Fig. 3A ).


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  		Table 1. GO analysis of the effects of CREB and CREM loss on transcription


Figure 3
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  		Figure 3. Disrupted cholesterol homeostasis in Creb1Camkcre4; Crem–/– neurons. A) Analysis of gene expression data points at a general dysregulation of sterol trafficking and metabolism. Genes critically involved in cholesterol homeostasis are listed. Green indicates decreased abundance of transcript; red indicates increased abundance. B–E) The loss of CREB and CREM leads to accumulation of cholesterol, as visualized by filipin staining. Striata of control (Creb1flox/flox; Crem+/–) animals (B) show only background fluorescence, due to membrane staining. In striata (C) or cortex (D) of mutant animals (Creb1Camkcre4; Crem–/–), a clear accumulation of cholesterol is visible. Confocal analysis of a double immunofluorescent staining of the Cre recombinase (green), which is exclusively expressed in neurons, and filipin (blue) shows that cholesterol accumulation occurs in the neurons (E). Original view: x210 (B, C); x300 (D); x640 (E).

Genes involved in the cholesterol biosynthesis pathway are controlled transcriptionally by a negative feedback loop that prevents their expression when cholesterol levels are high (29) . Thus, coordinated down-regulation of the pathway suggests that cells sense an excess of cholesterol. To test this hypothesis more directly and to gain cellular resolution on the alterations suggested by the computational analysis above, we stained brain sections with filipin, a fluorescent dye specifically associating with cholesterol. Staining of control animals gave rise to a general fluorescent background due to the staining of cellular membrane structures. In contrast, staining of sections from mutant animals revealed that neurons displayed a massive accumulation of intracellular cholesterol (Fig. 3B, C ). This phenotype could be observed in the hippocampus and the striatum and in scattered neurons in the cerebral cortex as well (Fig. 3B-E and data not shown). Thus, the expression signature detected in our microarray experiments led us to the identification of an unforeseen functional alteration in the CREB/CREM-deficient neurons, which appear to accumulate considerable levels of intracellular cholesterol.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We found that only a very specific set of activity-regulated genes is dependent on CREB and CREM for its induction in the hippocampus after neuronal activity induced by kainic acid. Given the enormous interest attributed to the role of CREB in hippocampal plasticity, the relatively low impact of its ablation on activity-regulated transcription is rather surprising. In particular, the finding that loss of CREB and CREM does not affect the inducibility of Fos is striking, as the promoter of this gene has been the most widely used model to reveal the role of CREB in activity-dependent transcription in vitro. Recent large-scale chromatin immunoprecipitation experiments estimate that the total number of CREB binding sites in the mammalian genome is 4000 to 30,000 (16 , 18) . Thus, it is clear from our data that CREB binds to a lot of regulatory gene regions without being critical for the expression of the gene. This likely means that several transcription factors collaborate in inducing the expression of most genes and that the removal of one factor is often not enough to cause any reduction of the expression. However, markedly reduced induction in the absence of CREB and CREM of a specific subset of activity-dependent genes, including Nr4a2, Gadd45b, Gpr19, and Tiparp, was observed.

Intriguingly, the fraction of CREB/CREM-dependent activity-regulated genes was much greater in the striatum after cocaine induction than in the hippocampus after kainic acid treatment. Thus, the induction of most activity-regulated genes found to be CREB/CREM independent in the hippocampus was severely attenuated in the striatum also. Several facts suggest that the abrogated induction was not due to apoptosis or a general defect of the neuronal circuitry. First, our in vitro data prove that CREB/CREM deficiency also attenuates immediate-early gene induction in a striatal cell line, showing that the CREB/CREM dependency is a cell-autonomous feature. In addition, the Creb1Camkcre4; Crem–/– mice show normal acute locomotor response to cocaine administration (not shown), indicating that cocaine also activates the striatum in mutant mice, but that there is a specific defect in the conversion of this activation into a transcriptional response.

The observed pattern of induced expression after cocaine or kainate treatment is in good agreement with previous reports, overlapping with immediate early genes identified in the hippocampus (1 , 30) and the striatum (31) . Our data were also consistent with the previously reported effects of binge-cocaine treatment on gene expression in the striatum (32) . Furthermore, a significant overlap was observed between transcripts we found induced in the control mice treated with kainate or cocaine and those previously reported by McClung and Nestler (22) to be up-regulated by CREB or {Delta}FosB overexpression in the nucleus accumbens. This was no longer the case for the profile of expression in the striatum after the loss of CREB and CREM, but the overlap did persist in the hippocampus despite the mutation.

One possible explanation for the differences in the role of CREB and CREM in the control of gene transcription between the striatum and hippocampus is that the set of transcription factors and coactivators present at the regulatory regions of a given gene, and their relative importance, may differ significantly between different cells and stimuli (33 , 34) . The pattern of CREB binding to regulatory regions in the genome may also differ between different cells and stimuli, as has been shown for SRF (35) . To what extent other transcription factors involved in activity-regulated gene expression also show this high context dependency remains to be elucidated. It is likely that although the role of CREB is very dependent on the specific context, some other stimulus-dependent transcription factors are more "hardwired" to their targets.

The alterations in expression of genes involved in cholesterol biosynthesis may stem from neuron-glia interactions. Thus, it is known that, in the brain, astrocyte-derived cholesterol is imported via lipoprotein transport by neurons and may play an important role in synaptogenesis (36 , 37) . CREB binding sites have been described in some cholesterol biosynthetic genes (i.e., Cyp51), which could explain why these genes are down-regulated in absence of CREB (38) . However, we could not observe significant alterations in Cyp51 gene expression level on Creb1 and Crem knockdown in ST14A cells (data not shown). The cholesterol accumulation revealed in our histological analysis rather suggests that repression of the biosynthetic pathway might be in fact a consequence of the elevated cholesterol levels, via the classical feedback loop controlling cholesterol synthesis (29) . It is tempting to speculate that the expression changes in genes involved in cholesterol trafficking may cause the cholesterol accumulation, which in turn may cause the repression of the synthetic pathway. However, it is at present difficult to disentangle the causes and consequences of the disturbed cholesterol homeostasis, and given the crucial role played by astrocytes in neuronal cholesterol metabolism (39) , it is possible that specifying the exact role of the CREB transcriptional pathway may require significant advances in the systems biology of astrocyte-neuron interactions.

In conclusion, our data show that CREB signaling is an important regulator of the transcriptional response to neuronal activity. Unexpectedly, this regulating role was very specific and dependent on the context, illustrating the flexibility of the CREB transcriptional pathway. The microarray analysis further provided an unbiased approach that enabled the discovery of metabolic disturbances in Creb1Camkcre4; Crem–/– mice. Alterations of cholesterol transport cause neurodegeneration in Niemann-Pick type C disease (40) , and modulation of cholesterol metabolism may have an effect on Alzheimer’s disease (41) and Huntington’s disease (42) . Our findings raise the intriguing possibility that the neurodegeneration observed in the absence of CREB and CREM may share mechanism with neurodegenerative disorders in which neurons succumb due to metabolic problems and pathological intracellular accumulation of lipids.


   ACKNOWLEDGMENTS
 
We thank M. Westphal and R. Klären for their expert technical assistance, Dr. C. Ittrich for help with statistical analysis of the microarray data, and Drs. M. Hergenhahn, M. Hollstein, and N. Gretz for providing access to the Affymetrix platform. We thank Dr. E. Cattaneo (Department of Pharmacological Sciences and Centre for Stem Cell Research, University of Milano, Milano, Italy) for providing the ST14A cell line. We thank Drs. A. Nordheim and R. Parlato for critically reading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (Germany), the Fonds der Chemischen Industrie, the European Community, the Bundesministerium für Bildung und Forschung (Germany), and the Alexander von Humboldt-Stiftung (to G.S.). D.E. was supported by the European Molecular Biology Organization, the Swedish Research Council-Medicine, and the Jeanssons, Åke Wiberg, Lars Hierta, and Fredrik and Ingrid Thuring foundations.


   FOOTNOTES
 
1 These authors contributed equally to this work.

2 Current address: European Molecular Biology Organization, 69117 Heidelberg, Germany.

Received for publication February 12, 2008. Accepted for publication March 20, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Cole, A. J., Saffen, D. W., Baraban, J. M., Worley, P. F. (1989) Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation. Nature 340,474-476[CrossRef][Medline]
  2. Greenberg, M. E., Ziff, E. B., Greene, L. A. (1986) Stimulation of neuronal acetylcholine receptors induces rapid gene transcription. Science 234,80-83[Abstract/Free Full Text]
  3. Morgan, J. I., Cohen, D. R., Hempstead, J. L., Curran, T. (1987) Mapping patterns of c-fos expression in the central nervous system after seizure. Science 237,192-197[Abstract/Free Full Text]
  4. Guzowski, J. F. (2002) Insights into immediate-early gene function in hippocampal memory consolidation using antisense oligonucleotide and fluorescent imaging approaches. Hippocampus 12,86-104[CrossRef][Medline]
  5. Nguyen, P. V., Abel, T., Kandel, E. R. (1994) Requirement of a critical period of transcription for induction of a late phase of LTP. Science 265,1104-1107[Abstract/Free Full Text]
  6. Dudai, Y. (2004) The neurobiology of consolidations, or, how stable is the engram?. Annu. Rev. Psychol. 55,51-86[CrossRef][Medline]
  7. West, A. E., Griffith, E. C., Greenberg, M. E. (2002) Regulation of transcription factors by neuronal activity. Nat. Rev. Neurosci. 3,921-931[CrossRef][Medline]
  8. Herdegen, T., Leah, J. D. (1998) Inducible and constitutive transcription factors in the mammalian nervous system: control of gene expression by Jun, Fos and Krox, and CREB/ATF proteins. Brain Res. Brain Res. Rev. 28,370-490[CrossRef][Medline]
  9. Hoeffler, J. P., Meyer, T. E., Yun, Y., Jameson, J. L., Habener, J. F. (1988) Cyclic AMP-responsive DNA-binding protein: structure based on a cloned placental cDNA. Science 242,1430-1433[Abstract/Free Full Text]
  10. Carlezon, W. A., Jr, Duman, R. S., Nestler, E. J. (2005) The many faces of CREB. Trends Neurosci. 28,436-445[CrossRef][Medline]
  11. Han, J. H., Kushner, S. A., Yiu, A. P., Cole, C. J., Matynia, A., Brown, R. A., Neve, R. L., Guzowski, J. F., Silva, A. J., Josselyn, S. A. (2007) Neuronal competition and selection during memory formation. Science 316,457-460[Abstract/Free Full Text]
  12. Mayr, B., Montminy, M. (2001) Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat. Rev. Mol. Cell Biol. 2,599-609[CrossRef][Medline]
  13. Bito, H., Deisseroth, K., Tsien, R. W. (1996) CREB phosphorylation and dephosphorylation: a Ca(2+)- and stimulus duration-dependent switch for hippocampal gene expression. Cell 87,1203-1214[CrossRef][Medline]
  14. Impey, S., Obrietan, K., Wong, S. T., Poser, S., Yano, S., Wayman, G., Deloulme, J. C., Chan, G., Storm, D. R. (1998) Cross talk between ERK and PKA is required for Ca2+ stimulation of CREB-dependent transcription and ERK nuclear translocation. Neuron 21,869-883[CrossRef][Medline]
  15. Sheng, M., Dougan, S. T., McFadden, G., Greenberg, M. E. (1988) Calcium and growth factor pathways of c-fos transcriptional activation require distinct upstream regulatory sequences. Mol. Cell. Biol. 8,2787-2796[Abstract/Free Full Text]
  16. Impey, S., McCorkle, S. R., Cha-Molstad, H., Dwyer, J. M., Yochum, G. S., Boss, J. M., McWeeney, S., Dunn, J. J., Mandel, G., Goodman, R. H. (2004) Defining the CREB regulon: a genome-wide analysis of transcription factor regulatory regions. Cell 119,1041-1054[Medline]
  17. Lonze, B. E., Ginty, D. D. (2002) Function and regulation of CREB family transcription factors in the nervous system. Neuron 35,605-623[CrossRef][Medline]
  18. Zhang, X., Odom, D. T., Koo, S. H., Conkright, M. D., Canettieri, G., Best, J., Chen, H., Jenner, R., Herbolsheimer, E., Jacobsen, E., Kadam, S., Ecker, J. R., Emerson, B., Hogenesch, J. B., Unterman, T., Young, R. A., 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[Abstract/Free Full Text]
  19. Bonni, A., Brunet, A., West, A. E., Datta, S. R., Takasu, M. A., Greenberg, M. E. (1999) Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science 286,1358-1362[Abstract/Free Full Text]
  20. Walton, M., Woodgate, A. M., Muravlev, A., Xu, R., During, M. J., Dragunow, M. (1999) CREB phosphorylation promotes nerve cell survival. J. Neurochem. 73,1836-1842[Medline]
  21. Mantamadiotis, T., Lemberger, T., Bleckmann, S. C., Kern, H., Kretz, O., Villalba, Martin A., Tronche, F., Kellendonk, C., Gau, D., Kapfhammer, J., Otto, C., Schmid, W., Schutz, G. (2002) Disruption of CREB function in brain leads to neurodegeneration. Nat. Genet. 31,47-54[CrossRef][Medline]
  22. McClung, C. A., Nestler, E. J. (2003) Regulation of gene expression and cocaine reward by CREB and DeltaFosB. Nat. Neurosci. 6,1208-1215[CrossRef][Medline]
  23. Blendy, J. A., Kaestner, K. H., Weinbauer, G. F., Nieschlag, E., Schutz, G. (1996) Severe impairment of spermatogenesis in mice lacking the CREM gene. Nature 380,162-165[CrossRef][Medline]
  24. Gentleman, R. C., Carey, V. J., Bates, D. M., Bolstad, B., Dettling, M., Dudoit, S., Ellis, B., Gautier, L., Ge, Y., Gentry, J., Hornik, K., Hothorn, T., Huber, W., Iacus, S., Irizarry, R., Leisch, F., Li, C., Maechler, M., Rossini, A. J., Sawitzki, G., Smith, C., Smyth, G., Tierney, L., Yang, J. Y., Zhang, J. (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5,R80[CrossRef][Medline]
  25. Subramanian, A., Tamayo, P., Mootha, V. K., Mukherjee, S., Ebert, B. L., Gillette, M. A., Paulovich, A., Pomeroy, S. L., Golub, T. R., Lander, E. S., Mesirov, J. P. (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. U. S. A. 102,15545-15550[Abstract/Free Full Text]
  26. Cattaneo, E., Conti, L. (1998) Generation and characterization of embryonic striatal conditionally immortalized ST14A cells. J. Neurosci. Res. 53,223-234[CrossRef][Medline]
  27. Hummler, E., Cole, T. J., Blendy, J. A., Ganss, R., Aguzzi, A., Schmid, W., Beermann, F., Schutz, G. (1994) Targeted mutation of the CREB gene: compensation within the CREB/ATF family of transcription factors. Proc. Natl. Acad. Sci. U. S. A. 91,5647-5651[Abstract/Free Full Text]
  28. Tang, Y., Lu, A., Aronow, B. J., Wagner, K. R., Sharp, F. R. (2002) Genomic responses of the brain to ischemic stroke, intracerebral haemorrhage, kainate seizures, hypoglycemia, and hypoxia. Eur. J. Neurosci. 15,1937-1952[CrossRef][Medline]
  29. Brown, M. S., Goldstein, J. L. (1997) The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89,331-340[CrossRef][Medline]
  30. Lanahan, A., Worley, P. (1998) Immediate-early genes and synaptic function. Neurobiol. Learn. Mem. 70,37-43[CrossRef][Medline]
  31. Berke, J. D., Paletzki, R. F., Aronson, G. J., Hyman, S. E., Gerfen, C. R. (1998) A complex program of striatal gene expression induced by dopaminergic stimulation. J. Neurosci. 18,5301-5310[Abstract/Free Full Text]
  32. Yuferov, V., Kroslak, T., Laforge, K. S., Zhou, Y., Ho, A., Kreek, M. J. (2003) Differential gene expression in the rat caudate putamen after "binge" cocaine administration: advantage of triplicate microarray analysis. Synapse 48,157-169[CrossRef][Medline]
  33. Ravnskjaer, K., Kester, H., Liu, Y., Zhang, X., Lee, D., Yates, J. R., 3rd, Montminy, M. (2007) Cooperative interactions between CBP and TORC2 confer selectivity to CREB target gene expression. EMBO J. 26,2880-2889[CrossRef][Medline]
  34. Xu, W., Kasper, L. H., Lerach, S., Jeevan, T., Brindle, P. K. (2007) Individual CREB-target genes dictate usage of distinct cAMP-responsive coactivation mechanisms. EMBO J. 26,2890-2903[CrossRef][Medline]
  35. Cooper, S. J., Trinklein, N. D., Nguyen, L., Myers, R. M. (2007) Serum response factor binding sites differ in three human cell types. Genome Res. 17,136-144[Abstract/Free Full Text]
  36. Funfschilling, U., Saher, G., Xiao, L., Mobius, W., Nave, K. A. (2007) Survival of adult neurons lacking cholesterol synthesis in vivo. BMC Neurosci. 8,1[CrossRef][Medline]
  37. Mauch, D. H., Nagler, K., Schumacher, S., Goritz, C., Muller, E. C., Otto, A., Pfrieger, F. W. (2001) CNS synaptogenesis promoted by glia-derived cholesterol. Science 294,1354-1357[Abstract/Free Full Text]
  38. Halder, S. K., Fink, M., Waterman, M. R., Rozman, D. (2002) A cAMP-responsive element binding site is essential for sterol regulation of the human lanosterol 14alpha-demethylase gene (CYP51). Mol. Endocrinol. 16,1853-1863[Abstract/Free Full Text]
  39. Fagan, A. M., Holtzman, D. M. (2000) Astrocyte lipoproteins, effects of apoE on neuronal function, and role of apoE in amyloid-beta deposition in vivo. Microsc. Res. Tech. 50,297-304[CrossRef][Medline]
  40. Loftus, S. K., Morris, J. A., Carstea, E. D., Gu, J. Z., Cummings, C., Brown, A., Ellison, J., Ohno, K., Rosenfeld, M. A., Tagle, D. A., Pentchev, P. G., Pavan, W. J. (1997) Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene. Science 277,232-235[Abstract/Free Full Text]
  41. Michikawa, M. (2004) Neurodegenerative disorders and cholesterol. Curr. Alzheimer Res. 1,271-275[CrossRef][Medline]
  42. Valenza, M., Carroll, J. B., Leoni, V., Bertram, L. N., Bjorkhem, I., Singaraja, R. R., Di Donato, S., Lutjohann, D., Hayden, M. R., Cattaneo, E. (2007) Cholesterol biosynthesis pathway is disturbed in YAC128 mice and is modulated by huntingtin mutation. Hum. Mol. Genet 16,2187-2198[Abstract/Free Full Text]



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