# THE TRANSCRIPTIONAL REGULATION OF MEMORY

EDITED BY: Benedict C. Albensi and Jelena Djordjevic PUBLISHED IN: Frontiers in Molecular Neuroscience

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ISSN 1664-8714 ISBN 978-2-88919-865-8 DOI 10.3389/978-2-88919-865-8

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# **THE TRANSCRIPTIONAL REGULATION OF MEMORY**

Topic Editors:

**Benedict C. Albensi,** University of Manitoba, Canada **Jelena Djordjevic,** University of Manitoba, Canada

Dissociated adult rat sensory neurons, maintained in culture and immunostained, showing beta-tubilin III associated cytoskeleton (red) and cAMP Responsive Element Binding Protein 1 (CREB1) in the nucleus(green).

Image courtesy of Mohammad Golam Sabbir, PhD, St. Boniface Hospital Research Centre, University of Manitoba, Canada.

The formation of various forms of memory involves a series of distinct cellular and molecular mechanisms, many of which are not fully understood. There are highly conserved pathways that are involved in learning, memory, and synaptic plasticity, which is the primary substrate for memory storage. The formation of short-term (across minutes) memory is mediated by local changes in synapses, while long-term (across hours to days) memory storage is associated with activation of transcription and synthesis of proteins that modify synaptic function. Transcription factors, which can either repress or activate transcription, play a vital role in driving protein synthesis underlying synaptic plasticity and memory, whereby protein synthesis provides the necessary building blocks to accommodate structural changes at the synapse that foster memory formation. Recent data implicate several families of transcription factors that appear critically important in the regulation of memory. In this Topic we will focus on the families of transcription factors thus far found to be critically involved in synaptic plasticity and memory formation. These include cAMP response element binding protein (CREB), Rel/nuclear factor B (Rel/NFB), CCAAT enhancer binding protein (C/EBP), and early growth response factor (Egr).

In recent years, numerous studies have implicated epigenetic mechanisms, changes in gene activity and expression that occur without alteration in gene sequence, in the memory consolidation process. DNA methylation and chromatin remodeling are critically involved in learning and memory, supporting a role of epigenetic mechanisms. Here we provide more evidence of the importance of DNA methylation, histone posttranslational modifications and the role of histone acetylation and HDAC inhibitors in above mentioned processes.

**Citation:** Albensi, B. C., Djordjevic, J., eds. (2016). The Transcriptional Regulation of Memory. Lausanne: Frontiers Media. doi: 10.3389/978-2-88919-865-8

# Table of Contents

# Editorial: Transcriptional Regulation of Memory

#### Benedict C. Albensi\* and Jelena Djordjevic

*Laboratory of Synaptic Plasticity and Memory Dysfunction, Division of Neurodegenerative Disorders, Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, MB, Canada*

#### Keywords: molecular, memory, transcription, epigenetic, synaptic plasticity (LTP/LTD)

#### **The Editorial on the Research Topic**

#### **Transcriptional Regulation of Memory**

Memory is the capacity for retaining and reviving information, facts, events, impressions, etc., and of recalling or recognizing previous experiences (Kandel et al., 2013). The formation of human memories involves a series of complicated biochemical processes, many of which are not fully understood. Short-term, intermediate-term, and long-term memories have different biological and molecular substrates and therefore represent distinct forms of memory. A short-term memory is the retention of information for a brief time (seconds to minutes) without creation of the neural changes for later recall. In contrast, a long-term memory (hours to weeks to years) occurs when changes in neural pathways result in the storage of information that can be recalled weeks, months, or even years later. There are thousands of molecules presumably involved in memory. However, recent data implicate several families of transcription factors and epigenetic processes that appear critical in the regulation of memory. Transcription factors are already well-known to regulate the basal process of transcription, the selective activation of genes, and/or the repression of genes. Albeit, we further acknowledge the importance of transcriptional processes for both memory consolidation and reconsolidation, and recognize there are still a lot of unanswered questions: For example, what are the key transcription factors involved in the regulation of memory? What are their gene targets and how do they mediate memory formation? How is transcription regulated over time? How does epigenetics contribute to memory? Can the context or our experience influence the pattern of transcription and regulate the formation of memory?

#### Edited and reviewed by:

*Jochen C. Meier, Technical University Braunschweig, Germany*

#### \*Correspondence:

*Benedict C. Albensi<EMAIL_ADDRESS>
Received: *22 February 2016* Accepted: *24 March 2016* Published: *19 April 2016*

#### Citation:

*Albensi BC and Djordjevic J (2016) Editorial: Transcriptional Regulation of Memory. Front. Mol. Neurosci. 9:24. doi: 10.3389/fnmol.2016.00024*

To this end, emerging from the literature are examples of transcription factors, transcriptional processes, mechanisms of epigenetics that appear to be critically involved in synaptic plasticity and long term memory. Some of these include histone variant H3.3, nuclear factor kappa B (NF-kB) protein, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein, histone deacetylase inhibitors, Gadd45beta expression, CREB-regulated transcriptional coactivator (CRTC1), CREB, and early growth response (Egr) factor protein. The existence of genetic interactions, epigenetic processes, and non-genetic mechanisms that transfer unique environmental context to the memory formation is of growing interest.

This Frontiers in Molecular Neuroscience Research Topic-Special Issue on the Transcriptional Regulation of Memory includes both reviews and experimental studies focused on transcription factors and epigenetic processes that regulate various forms of memory, sometimes in unique ways. Dr. Ted Abel and colleagues (McNally, et al.) developed a transgenic mouse line that expresses a tetO-regulated, hemagglutin (HA)-tagged histone H3.3, which they demonstrate has control over transgene expression. In this study, they characterize this mouse line during memory consolidation and evaluate the contribution of H3.3 to fear memory and motor learning. Drs. Barbra Kaltschmidt and Christian Kaltschmidt (Kaltschmidt and Kaltschmidt) discuss roles for NF-kB in structural plasticity and long-term memory. In this review, they also discuss differences in NF-kB activity in

neurons versus glia related to NF-kB homeostasis and whether NF-kB activation is a friend or foe. Data from my laboratory is also presented by Dr. Snow (Snow et al.) that demonstrates training in the Morris water maze (MWM) is associated with the upregulation of the NF-kB p65 subunit, correlations with CREB, increases in the factor Nrf2, and elevations of actin protein. These data show that the MWM is associated with transcriptional changes in the hippocampus, including those related to redox regulation. Dr. Feng and colleagues (Jia et al.) go on to discuss epigenetic regulation and how the dysregulation of histone acetylation contributes to postoperative cognitive dysfunction. In this study, the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA) was shown to attenuate long term memory impairments in aging mice. Dr. Farah Lubin and colleagues (Jarome et al.) also present a study on epigenetic regulation in memory consolidation. In this case, they demonstrate a novel role for NF-kB in the regulation of Gadd45beta expression and DNA methylation during fear memory formation. Dr. Arturo Romano and his colleagues (de la Fuente et al.) also focus on NF-kB activity in their review and specifically address consolidation and reconsolidation memory phases. In addition, there is a discussion on the regulation of immediate-early and late genes by epigenetic mechanisms that determine enduring forms of memories. Moving on to a more classically recognized transcription factor pathway, Dr. Kelsey Martin and colleagues (Ch'ng et al.) present a study on the CREB-regulated transcriptional coactivator (CRTC1) in regulating learning and memory. Here they provide evidence for the sources of calcium required for the nuclear import of CRTC1 and show CRTC1 undergoes dynein-mediated and nuclear localization signal (NLS)-dependent transport along microtubules to reach the nucleus. Dr. Sylvia Ortega-Martínez also presents a paper on CREB that reviews various members of the CREB family. The review also highlights the role of CREB as a modulator of adult hippocampal neurogenesis (AHN) during memory consolidation. Finally, Dr. Joanna Williams

#### and her colleagues (Nido et al.) present a study on the genomic response following long-term potentiation (LTP), an experimental paradigm associated with memory encoding. These data present the view that a new homeostatic state is created 24 h post LTP, by which the stability of neural networks regulated at different times parallel properties observed at the synapse.

From reading these papers, it becomes obvious that the formation of human memories involves a series of complicated biochemical processes many of which are not fully understood. The studies and reviews presented here implicate several families of transcription factors and epigenetic mechanisms that highlight the complexity associated with the transcriptional regulation of memory.

Finally we wish to thank the Frontiers family of open access journals for their excellent support in putting this series of reviews and studies together for this Research Topic.

# AUTHOR CONTRIBUTIONS

All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.

# FUNDING

St. Boniface Hospital Research Foundation, Alzheimer Society of Manitoba, Natural Sciences and Engineering Council of Canada (NSERC), and Research Manitoba.

# ACKNOWLEDGMENTS

BA is the Manitoba Dementia Research Chair and the Honourable Douglas and Patricia Everett and Royal Canadian Properties Limited Endowment Fund Chair. He is also a Research Affiliate of the Centre on Aging at the University of Manitoba and a Scientist at the Children's Hospital Research Institute of Manitoba.

# REFERENCES

Kandel, E. R., Schwartz, J. H., Jessell, T. M., Siegelbaum, S. A., and Hudspeth, A. J., (2013). Principles of Neural Science, 5th Edn. McGraw-Hill Companies.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Albensi and Djordjevic. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# NF-κB transcription factor role in consolidation and reconsolidation of persistent memories

Verónica de la Fuente, Noel Federman, Gisela Zalcman, Angeles Salles, Ramiro Freudenthal and Arturo Romano\*

Laboratorio de Neurobiología de la Memoria, Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE, UBA-CONICET), Universidad de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina

Transcriptional regulation is an important molecular process required for long-term neural plasticity and long-term memory (LTM) formation. Thus, one main interest in molecular neuroscience in the last decades has been the identification of transcription factors that are involved in memory processes. Among them, the nuclear factor κB (NF-κB) family of transcription factors has gained interest due to a significant body of evidence that supports a key role of these proteins in synaptic plasticity and memory. In recent years, the interest was particularly reinforced because NF-κB was characterized as an important regulator of synaptogenesis. This function may be explained by its participation in synapse to nucleus communication, as well as a possible local role at the synapse. This review provides an overview of experimental work obtained in the last years, showing the essential role of this transcription factor in memory processes in different learning tasks in mammals. We focus the review on the consolidation and reconsolidation memory phases as well as on the regulation of immediate-early and late genes by epigenetic mechanisms that determine enduring forms of memories.

#### Keywords: NF-κB, transcription factors, consolidation, reconsolidation, memory

#### Transcription Factors and Memory

Early in the research on the molecular basis of memory it was proposed that memory consolidation is a period during which new proteins must be synthesized (Davis and Squire, 1984; Goelet et al., 1986). The first evidence came from the use of antibiotics like puromycin and cycloheximide (Flexner et al., 1963; Agranoff and Klinger, 1964; Barondes and Cohen, 1966, 1967), which are protein synthesis inhibitors. These inhibitors were effective when administered shortly before or after acquisition, and induced memory impairment only when tested at long-term but not at short-term periods. Almost at the same time of these findings with protein synthesis inhibitors, the role of mRNA synthesis in memory formation was initially evidenced by the use of the antibiotic actinomycin D, which interferes with transcription (Barondes and Jarvik, 1964; Appel, 1965; Cohen and Barondes, 1966). Like the other antibiotics, actinomycin D impaired long-term memory (LTM) only when administered around training session, and the amnesic effect was manifested when tested at long-term periods. Many years later, the first transcription factors involved in synaptic plasticity, cAMP response element binding protein (CREB; Dash et al., 1990) and in memory consolidation, c-Fos (a member of the activated protein 1-AP-1-family; Anokhin and Rose, 1991) were identified. Subsequent work on CREB, from a series

#### Edited by:

Benedict C. Albensi, University of Manitoba, Canada

#### Reviewed by:

Hansjürgen Volkmer, NMI an der Universität Tübingen, Germany Lan Ma, Fudan University, China

#### \*Correspondence:

Arturo Romano, Laboratorio de Neurobiología de la Memoria, Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE, UBA-CONICET), Universidad de Buenos Aires, Ciudad Universitaria, Pab. II, 2do Piso, 1428EGA Buenos Aires, Argentina<EMAIL_ADDRESS>
> Received: 29 May 2015 Accepted: 24 August 2015 Published: 09 September 2015

#### Citation:

de la Fuente V, Federman N, Zalcman G, Salles A, Freudenthal R and Romano A (2015) NF-κB transcription factor role in consolidation and reconsolidation of persistent memories. Front. Mol. Neurosci. 8:50. doi: 10.3389/fnmol.2015.00050 of studies in Aplysia, Drosophila and rodents, supported that this transcription factor takes part in an evolutionarily conserved molecular mechanism involved in neural plasticity associated with memory formation (Kaang et al., 1993; Bourtchuladze et al., 1994; Yin et al., 1994).

The identification of other transcription factors involved in neural plasticity and memory was achieved in the following years, such as CAAT enhancer binding protein (C/EBP; Alberini et al., 1994), zinc finger inducing factor 268 (Zif268; Okuno and Miyashita, 1996; Jones et al., 2001) and nuclear factor κB (NF-κB; Freudenthal et al., 1998; Freudenthal and Romano, 2000). For these five transcription factors, strong evidence is now available supporting their essential function in memory processes (Alberini, 2009).

# NF-κB Transcription Factor in the Nervous System

NF-κB was first characterized in lymphocytes but is ubiquitously expressed in different tissues. In mammals, the Rel/NF-κB family of transcription factors consists of five members: p50, a product of the NF-κB1 gene; p52, a product of the NF-κB2 gene; p65 or RelA; c-Rel; and RelB. The Rel/NF-κB family also shares the Rel homology domain containing the following structural regions: DNA binding, dimerization, nuclear localization, and inhibitor κB (IκB) interaction sites. Members of the Rel/NF-κB family bind to DNA, recognizing the consensus decameric sequence 5 0 -GGGPuNNPyPyCC-3<sup>0</sup> , designated as the κB site. Different dimers bind with diverse affinities to the distinct κB sites. In addition, gene transcription can be activated or repressed depending on the dimer composition. For example, heterodimers between Rel and NF-κB 1 or NF-κB 2 are activators while homodimers of NF-κB 1 and NF-κB 2 are repressors. Evidence for the existence of Rel/NF-κB members has been found in different species such as humans, rats, mice, chicks, Xenopus, Drosophila, Anopheles, honeybees, crabs, oysters and Aplysia, indicating that this transcription factor family is evolutionarily conserved. At variance with most of the transcription factors that are normally found in the nucleus, inactive NF-κB dimers are usually retained in the cytoplasm by binding to IκB inhibitory protein. IκB occludes the nuclear localization signal (NLS) of NF-κB subunits (Hayden and Ghosh, 2008). Upon stimulation, the IκB kinase complex becomes activated and phosphorylates IκB, leading to its ubiquitination and subsequent degradation. In the canonical pathways for NF-κB activation, this degradation allows NF-κB to translocate to the nucleus and regulate the expression of its target genes. NF-κB is expressed in the nervous system both in neurons and non neuronal cells such as glia. The subunits most commonly expressed in neurons are p50-p65 heterodimers and p50 homodimers. NF-κB has been found in pre- and postsynaptic sites (Kaltschmidt et al., 1993; Guerrini et al., 1995; Meberg et al., 1996; Freudenthal and Romano, 2000). The presence of inducible NF-κB in synapses led to the hypothesis that NF-κB is locally activated by synaptic activity and then, in this activated state, is retrogradely transported to the nucleus to regulate gene expression. This fact would confer to this transcription factor a dual function as synaptic activity detector and as transcriptional regulator.

Until now, a number of NF-κB target genes have been found in neurons, and many of them are related to neural plasticity and memory. These include the genes that codify for the following proteins: calcium/calmodulin protein kinase II delta (CaMKIIδ) (see below, Federman et al., 2013), insulinlike growth factor 2 (IGF2; Schmeisser et al., 2012), Zif268 immediate-early gene (Zhou et al., 2003), C/EBP delta (Liu et al., 2007), brain-derived neurotrophic factor (Saha et al., 2006), neural cell adhesion molecule (NCAM; Simpson and Morris, 2000), inducible and neural nitric oxide synthetase (NOS; Morris et al., 2003), cAMP dependent protein kinase A (PKA) α catalytic subunit (Kaltschmidt et al., 2006), protein kinase C (PKC) δ (Suh et al., 2003), and amyloid precursor protein (APP; Song and Lahiri, 1998). NF-κB is widely distributed in the central nervous system (CNS) in regions such as the hippocampus, cerebral cortex, and cerebellum. It is activated upon glutamatergic excitatory synaptic transmission by signals involved in neural plasticity such as depolarization and transient increment of intracellular Ca2<sup>+</sup> through the Ca2+-dependent signaling and the activation of CaMKII (Meffert et al., 2003). NF-κB is also activated in response to dopamine and by neuropetides such as angiotensin II (Frenkel et al., 2002) and by NCAM (Krushel et al., 1999).

# NF-κB in Neural Plasticity and Memory

Long-term potentiation (LTP) and long-term depression (LTD) are models of synaptic plasticity that increase or decrease, respectively, the synaptic efficacy. The induction of in vivo LTP in hippocampus increased the expression and the activation of NF-κB (Meberg et al., 1996; Freudenthal et al., 2004). Accordingly, the inhibition of NF-κB reduced LTP and impaired LTD (Albensi and Mattson, 2000). Thus, NF-κB is required in these experimental processes of synaptic plasticity which are considered good models of the synaptic changes occurring during memory storage.

The first evidence of the involvement of NF-κB in memory was obtained in invertebrates, in the context-signal memory of the crab Chasmagnathus. In this model, in which contextual cues are associated with a danger stimulus, the activation of NF-κB highly correlates with LTM formation (Freudenthal et al., 1998; Freudenthal and Romano, 2000) while inhibition of this transcription factor impairs memory formation (Merlo et al., 2002). Further studies found NF-κB involved in memory processes in rodents, in different learning tasks such as fear startle potentiation (Yeh et al., 2002), Inhibitory Avoidance (IA; Freudenthal et al., 2005), radial arms maze (Meffert et al., 2003), contextual fear conditioning (FC; Lubin and Sweatt, 2007), conditioned place preference (Yang et al., 2011) and novel object recognition (NOR; Federman et al., 2013).

In the following sections, we will review a series of recent studies in rodents describing the involvement of NF-κB, not only in the memory formation, but also in the different phases of memory, its persistence and its role at the synapse.

# NF-κB in Memory Consolidation

Initially, evidence for the role of Rel/NF-κB in rodents was obtained studying fear-potentiated startle conditioning in rats (Yeh et al., 2002). In this task, trained animals received 10 light stimuli paired with a footshock, and controls received a pseudorandom presentation of the same number of light stimuli and footshocks. In a testing session, the conditioned stimulus was presented together with a white noise and the fearpotentiated startle was evaluated. Using this model, an increment of NF-κB DNA-binding activity was found by electrophoretic mobility shift assay at 2–6 h after training in amygdala but not in hippocampus or cerebellum. IKK activation was also found at 10 and 30 min after training, decreasing to basal levels at 90 min. The intra-amygdala inhibition of NF-κB before training significantly reduced potentiated startle at LTM testing, supporting the idea that NF-κB activation in amygdala is required for memory storage. A further study in this model revealed that FC induced an association between NF-κB and the acetyl transferase CREB binding protein (CBP), resulting in an increase in acetylated p65. The administration of general deacetylase inhibitors prolonged the acetylation state of p65 in amygdala and facilitated LTM (Yeh et al., 2004).

The first evidence for the role of Rel/NF-κB in hippocampus was found in an IA task in mice (**Figure 1A**). The hippocampal formation is a key structure for contextual memories because it is involved in processing and identification of contextual characteristics of different places and in the coding of the unconditioned stimulus associated with a particular place. In this task, animals were placed on an illuminated platform in front of the entrance to a dark compartment. Once the animals entered the dark compartment, they received a mild footshock while controls did not. At LTM testing, trained animals typically showed high latencies to step-through or avoided entering the compartment. Conversely, control animals showed very low latencies to enter. Moreover, we found that the inhibition of this transcription factor by immediate posttraining i.c.v. administration of the IKK inhibitor sulfasalazine (SSZ) induced retention deficit in a dose-dependent manner when tested 48 h later (Freudenthal et al., 2005). On the contrary, delayed injections of SSZ at 3 or 24 h post-training did not affect retention. In order to study the effect of direct NF-κB inhibition, we used the κB decoy strategy. The κB decoy consists of a double-stranded DNA oligodeoxinucleotide (ODN) containing a κB consensus sequence that binds to the transcription factor, impeding its action. The κB decoy was administered i.c.v. 2 h pretraining, showing memory impairment. Conversely, injection of the κB decoy with a single base mutation did not affect LTM. Hippocampal NF-κB activity after training increased in shocked animals at 45 min, having returned to basal levels at 2 h post training. These results support the idea that NF-κB activation in the hippocampus is required for the consolidation of contextual features that constitute the conditioned stimulus representation (Freudenthal et al., 2005).

We also studied the dynamics of hippocampal NF-κB activation in the consolidation of contextual fear memory (**Figure 1A**). In this task, mice associate contextual characteristics of the training chamber with a mild footshock. During testing, the percentage of freezing response is determined. Similar to the results obtained in IA, an activation of hippocampal NF-κB at 45 min was observed after the three-trials training (de la Fuente et al., 2014). The intra-hippocampal administration of SSZ induced amnesic effects, which were revealed by further testing sessions at 24 h and 2 weeks after training (de la Fuente et al., 2014), suggesting a permanent amnesic effect. These findings support that NF-κB is involved in contextual fear memory consolidation in the hippocampus.

IA and FC both entail the presence of an aversive reinforcement, the mild footshock that acts as unconditioned stimulus. At this point, one possible interpretation that could be formulated is that NF-κB is specifically involved in memories associated with aversive and stressful stimuli. NOR is a widely investigated form of declarative memory. It refers to the ability of determining whether a newly found item or object has been previously encountered (Squire et al., 2007). In the classical variant of the task, the animal is exposed during training to two identical objects and is allowed to explore them for a defined time period. During testing, animals are exposed to two different objects, one of them is identical to the training objects and the other is a novel object. The time of exploration of the two objects is then determined to obtain a discrimination index, taking advantage of the rodent's natural predisposition to explore novel objects more thoroughly. In the NOR, on the one hand, aversive stimuli are absent but on the other hand, the involvement of the hippocampus in this task is a matter of debate (Warburton and Brown, 2010). We then tested whether NF-κB is activated in hippocampus during NOR memory consolidation and found that this transcription factor is active in the nucleus of hippocampal cells at both 45 and 60 min. In other experiment, the infusion of κB decoy in the dorsal hippocampus after training drastically impaired object discrimination when tested 24 h post training (Federman et al., 2013; Zalcman et al., 2015; **Figure 1C**). All in all, these experiments support both the participation of NF-κB in NOR memory consolidation and the involvement of the hippocampus in this task.

Transcription factors such as NF-κB are involved in the first step of gene expression regulation. These factors are already present at the moment of neuronal activity induced by training. The activation of this kind of transcription factors allows for a second step in gene regulation by induction of immediate-early genes (IEGs) whose protein products are, in turn, transcription factors. Among these IEGs, zif268 has been found to play a critical role in LTM formation and reprocessing after retrieval. NF-κB binding to the zif268 promoter region has been identified (Thyss et al., 2005), supporting that NF-κB is involved in its expression. We recently found that Zif268 protein expression was induced 45 min after NOR training in the hippocampus and that the post-training intrahippocampal administration of ODN antisense of the Zif268 mRNA impaired recognition memory. In addition, we found that NF-κB inhibition by κB decoy ODN significantly reduced the traininginduced Zif268 increment, indicating that NF-κB is involved in the regulation of Zif268 expression in the hippocampus

during recognition memory consolidation (Zalcman et al., 2015; **Figure 1C**).

# NF-κB in Memory Reconsolidation

In the classical postulation, memory is stored in its definitive long-term form once consolidated few hours after acquisition. During this process, memory becomes resistant to different disruption treatments. However, an important body of evidence has challenged this assumption, supporting the view that memory is more dynamic and can be modified when reactivated by a reminder during retrieval. Under these circumstances, a new period of lability followed by a consolidation-like process takes place. This phenomenon, known in literature as reconsolidation, has been under intense research for the last 15 years (Dudai, 2012).

In an initial report in mammals on the role of NF-κB in memory reconsolidation, we found that NF-κB inhibition in the hippocampus by SSZ or κB decoy after memory reactivation impaired retention of IA memory in mice (**Figure 1D**). In contrast, mutated κB decoy had no effect. Furthermore, we found NF-κB activation in the hippocampus, with a peak 15 min after memory retrieval (Boccia et al., 2007). This activation was faster than the one previously found in the hippocampus during consolidation of the same task, with a peak at 45 min (Freudenthal et al., 2005), suggesting a temporal signature for both processes. In contextual FC, when animals are reexposed for 5 min to the training context 1 day after training (once memory consolidation is fully achieved), a labilizationreconsolidation process takes place. During this process, we found that after an initial inhibition, NF-κB showed a peak of activation in mice hippocampus 15 min after the animal was removed from the context (**Figure 1D**), which is the same time point at which NF-κB was activated after retrieval in the IA task. The infusion of κB decoy immediately after a brief reexposure caused memory impairment when animals were tested a day after re-exposure, but not 4 h after re-exposure or when mice were not re-exposed. The memory deficit was present even when the mice were tested 2 weeks later, supporting a permanent impairment of memory (de la Fuente et al., 2011). All these findings led us to postulate that NF-κB is activated after memory reactivation as part of the molecular mechanisms involved in memory reconsolidation. Furthermore, the intra-hippocampal administration of the calcineurin phosphatase inhibitor FK506 previous to the re-exposure session induced NF-κB activation and a concomitant memory enhancement that was reverted by the administration of SSZ. Calcineurin actually acts constraining memory in both consolidation and reconsolidation by NF-κB inhibition (de la Fuente et al., 2014).

Lubin and Sweatt (2007) also found evidence on the role of the hippocampal NF-κB pathway in FC reconsolidation in rats, supporting the direct involvement of IKK, the protein kinase that activates NF-κB, in histone phosphorilation in the nucleus. In a recent study, Si et al. (2012) demonstrated that inhibition of NF-κB after memory reactivation in the basolateral amygdala impairs the retention of cued auditory FC. This latter finding supports that NF-κB is not only acting in reconsolidation in the hippocampus but also in amygdala for hippocampal independent tasks. Memory impairment by NF-κB inhibition in reconsolidation was also found in the morphine-induced conditioned place preference task (Yang et al., 2011). Thus, the role of NF-κB in memory reconsolidation is supported in different tasks and different areas of the brain.

# Knock-Out Mice for NF-κB Pathway

The use of gene-targeting strategies in mice gave further support to the role of NF-κB in learning and memory. Mice with a null mutation of p50 showed acquisition deficits in an active avoidance task (Kassed et al., 2002) but, strikingly, normal LTM 24 h after training. Similarly, in another report short-term but not LTM deficits were found in p50 deficient mice (Denis-Donini et al., 2008). Although these results would suggest that p50 is not required for LTM formation, the general effect of the p50 knock-out on κB-dependent transcription is not easy to predict, as this subunit is a component of both a transcriptional activator (p65/p50 heterodimer) and a repressor (p50/p50 homodimer). However, more recent studies of p50 knock-out mice showed that the absence of this NF-κB subunit produced late phase LTP impairment but not early phase LTP impairment, and deficits in long-term spatial memory in the Morris water maze. These results support the hypothesis that the p50 subunit is required in long-term plasticity and spatial memory in the hippocampus (Oikawa et al., 2012).

Baltimore and colleagues have systematically deleted individual NF-κB subunits in transgenic mice. Deletion of the p65 subunit is lethal during embryogenesis (Beg et al., 1995) but the knock-out can be rescued by concurrent deletion of type 1 TNF receptors (Alcamo et al., 2001). The brains of p65/TNFR1 double knock-out mice appear normal under general examination and no obvious behavioral abnormalities were described. With this mutant, Meffert et al. (2003) analyzed memory performance using the radial-arm maze with both the spatial and non-spatial (cued) version. In the spatial version of the task, the external end of all the eight arms had food pellets and spatial clues external to the maze were available. In order to optimize food rewards mice had to remember and to avoid the already visited arms. p65/TNFR1 KO mutants made more errors than p65+/+/TNFR1−/<sup>−</sup> siblings during the first sessions, but in the last sessions no differences in the level of errors were found. In the non-spatial cued version of the task, no external clues were available and only the arms that were illuminated had food pellets. No performance deficit was found in the cued version. The spatial version of this paradigm depends on hippocampus whereas this area is not involved in the non-spatial version. Thus, this result suggests that p65 is required in the hippocampus for neural plasticity necessary in spatial information storage.

Levenson et al. (2004) studied the effect of genetic deletion of c-Rel, a member of the NF-κB family, in FC. In the cued version, animals received a white noise paired with a foot-shock in the training chamber. During testing, freezing response to the noise was evaluated in a different chamber. No differences between c-Rel knock-out and wild type mice were found. However, in the contextual version of the task, in which the training chamber was paired with foot-shock, and the testing was performed in the same chamber, the c-Rel knock-out mice showed impaired LTM. Therefore, c-Rel protein seemed to be necessary for the contextual FC, which is hippocampal dependent, and not required for the cued version in which the hippocampus is not necessary. In a more recent work, the same laboratory found that c-rel−/<sup>−</sup> mice displayed significant deficits in freezing behavior 24 h after training for contextual FC, but showed normal freezing behavior in cued FC and in short-term contextual FC. Moreover, c-rel−/<sup>−</sup> mice showed impairment in NOR task. These results indicate that c-rel−/<sup>−</sup> mice have impaired hippocampusdependent memory formation (Ahn et al., 2008).

In another study, Kaltschmidt et al. (2006) generated a double transgenic mouse with the expression of tetracycline transactivator (tTA) under the CamKIIa promoter, which confers postnatal expression restricted to the forebrain. The second transgene includes a TetO promoter, regulated by tTA, which allows expression of an IκB super-repressor (nonphosphorylatable form of IκBα). This genetic manipulation caused a general inhibition of NF-κB in forebrain neurons. Treatment with doxycyline rescues the NF-κB deficiency. The performance of this double transgenic mouse without doxycycline (NF-κB deficient) was tested in the hippocampal dependent Morris water maze. The double transgenic animals showed higher latencies during training to reach to the platform than controls, and a deficit in the transfer test without the platform, in which the time spent searching in the correct quadrant was determined. However, no differences were found between these NF-κB-deficient animals and doxycyclinetreated animals (NF-κB-rescued), probably due to a nonspecific influence of the drug on memory formation. Besides the behavioral experiments, authors also showed that LTP and LTD were reduced in the Schaffer-collaterals hippocampal path. Notably, they found a down-regulation of the junD gene and the N-CAM gene in these double transgenic mice. In this same study, gene profiling experiments and analysis of regulatory regions identified the α catalytic subunit of PKA as a NF-κB target gene (Kaltschmidt et al., 2006).

In conclusion, most of the studies on genetic manipulation of the NF-κB pathway reviewed here point to an important role of this transcription factor in memory, particularly in hippocampaldependent tasks.

# Epigenetic Regulation of Persistent Memories

Epigenetic mechanisms regulate the magnitude and the extent of the gene expression pattern induced by learning. The genome of all cells is packaged in the chromatin, a structure comprising DNA and associated proteins, the histones, which determine the different degrees of DNA compaction in its basic structure, the nucleosome. Epigenetic marks consist of modifications of the chromatin structure, which affects transcription of genes. These marks may be post-translational modifications of histones, such as acetylation, phosphorylation, ubiquitination and methylation, as well as changes in the methylation patterns of DNA cytosine residues. Other epigenetic mechanisms include histone variants incorporation to nucleosomes, nucleosome remodeling, and changes in the position of the chromosome in relation to pores in the nuclear envelope (Raisner and Madhani, 2006; Draker and Cheung, 2009; Kundu and Peterson, 2009). All these epigenetic processes occur in an interdependent and coordinated manner, in order to regulate the expression of different parts of the genome (Mehler, 2008).

Previous studies showed that epigenetic mechanisms are involved in memory formation and in particular, in recognition memory. The histone acetyl transferase (HAT) CREB-binding protein (CBP)/p300 is involved in recognition memory consolidation (Oliveira et al., 2011). For instance, the HAT activity domain of CBP is required for NOR memory consolidation (Korzus et al., 2004; Wood et al., 2005) and knock-out mice of the cbp gene show deficits in the NOR task (Alarcón et al., 2004). Furthermore, by the use of specific drugs, it has been found that p300 activity is required for memory formation (Marek et al., 2011; Oliveira et al., 2011; Federman et al., 2013). As was previously mentioned, NF-κB is able to recruit CBP/p300 and to induce histone acetylation in promoter regions containing κB sites (Pradhan et al., 2012).

We hypothesized that NF-κB-dependent histone acetylation during memory consolidation is a molecular feature particularly required for the formation of enduring memories. To test this hypothesis we used three different training protocols: one group of animals received a weak training (3 min of object exploration) which did not induce LTM; another group received a standard training (10 min) that induced LTM which lasts for 24 h but not for 1 week; and the last group received a strong training (15 min) that induced LTM that lasts for 7 days. We found an induction of general histone H3 acetylation of the chromatin in the hippocampus only in animals trained with the strong training protocol. Therefore, these data suggest that the consolidation of more persistent memories differ at the molecular level from the less enduring memories by the induction of this epigenetic mechanism. Next, we explored if the increment in histone acetylation is dependent on NF-κB (**Figure 1E**). We found that NF-κB inhibition by κB decoy impaired memory persistence and concomitantly, prevented the induction of general H3 acetylation (Federman et al., 2013).

Following up on these findings, we investigated if specific genes involved in memory consolidation showed histone acetylation changes. In particular, we studied the promoters of zif268 as an instance of immediate-early gene (Davis et al., 2003; Soulé et al., 2008) and calcium/calmodulin kinase II δ (camk2d) as an instance of late gene (Sirri et al., 2010; Lucchesi et al., 2011). We found a significant increase in histone H3 acetylation at a specific NF-κB-regulated promoter region of the camk2d gene, which was reversed by NF-κB inhibition (**Figure 1E**). This H3 acetylation increment led to CaMKIIδ mRNA induction 3 h after strong training, but not after weaker training protocols (Federman et al., 2013). No changes in histone acetylation were found in the promoter of the immediate-early gene zif268. This work presents a molecular link between NF-κB transcription factor activation, epigenetic mechanisms, and late gene expression in the regulation of memory persistence.

# NF-κB in Neural Synaptic Plasticity

In the last years, important experimental results point to a key role of NF-κB in the determination of synaptogenesis (Boersma et al., 2011; Schmeisser et al., 2012), a basic structural process in the formation of the memory trace. In relation with these findings, the IGF2 gene has been recently identified as a novel IKK/NF-κB target. In IKK/NF-κB signaling-deficient neurons, synapse density is reduced and exogenous IGF2 is able to restore synapse density and promote spine maturation within 24 h. This process depends on IGF2 receptor via MEK/ERK activation. These findings illustrate a fundamental role of IKK/NF-κB–IGF2–IGF2R signaling in synapse formation and maturation in adult mice (Schmeisser et al., 2012). In fact, IGF2 has been previously identified as a memory enhancement factor (Chen et al., 2011).

Several results show that NF-κB is present at the synapses (Kaltschmidt et al., 1993; Guerrini et al., 1995; Meberg et al., 1996; Suzuki et al., 1997; Heckscher et al., 2007) and more recently, the role of the transcription factor during memory consolidation at this neuronal compartment has been explored. There is evidence supporting two major hypotheses regarding the transcription factor at this localization. The first one being that the transcription factor plays a role in the synapse-tonucleus communication, ultimately regulating gene expression (Wellmann et al., 2001). This synapse to nucleus transport is initiated by an increase in calcium that follows the N-Methyl-Daspartate (NMDA) receptor activation at the synapse (Wellmann et al., 2001; Meffert et al., 2003). A different hypothesis that could work simultaneously with the previous one, is that NF-κB may modulate synaptic function locally (Heckscher et al., 2007; Salles et al., 2014). On the one hand, it has been reported that the dynein/dynactin motor complex mediates the transport of NF-κB to the nucleus along microtubules and that this transport is dependent on an intact NLS (Mikenberg et al., 2007). It was also observed that NLSs from p50 and p65 are recognized by α importins and that such interaction is necessary for the translocation of the transcription factor to the nucleus (Fagerlund et al., 2005). It would be interesting to study the relation of importins with the dynein/dynactin motor complex in the transport/translocation of NF-κB to nucleus. On the other hand, and supporting the second hypothesis, we found that there are two pools of the transcription factor at this site, one free in the synaptoplasm and another one strongly bound to synaptic membranes (Salles et al., 2015; **Figure 1B**). The quantities of NF-κB for each pool change after training, decreasing in the synaptosomal content and increasing at the membranes 5 min post-training. This supports the idea that the free NF-κB may be either retrogradely transported to the nucleus or be translocated to synaptic membranes. Furthermore, we found that synaptosomal NF-κB activation occurs 5 min post-training during consolidation (Salles et al., 2015). Therefore synaptosomal activation has a different time course than nuclear activation (**Figure 1B**). This local activation and membrane dynamics support the idea that NF-κB may be important in the synaptic environment during consolidation. As outlined before, NF-κB may be involved in post-translational modifications of other proteins such as acetylation. There is at least one acetyl-transferase that has been described in dendrites and has been reported to interact with NF-κB, ADP-ribosylation factor domain protein 1 (ARD1; Ohkawa et al., 2008). It has been proposed that this protein is implicated in the microtubule dynamics during spine remodeling, suggesting an involvement of NF-κB in this process. Moreover, in the fruit fly neuromuscular

#### References

junction, NF-κB homolog Dorsal mediates the membrane insertion of glutamate receptors locally at a post-translational level (Heckscher et al., 2007). All this evidence points towards the idea that parallel to the retrograde transport of NF-κB to the nucleus, the transcription factor may play a key role at the synapses during memory consolidation.

#### Concluding Remarks

More than a decade after the initial descriptions of NF-κB's involvement in memory both in invertebrates (Freudenthal et al., 1998; Freudenthal and Romano, 2000) and vertebrates (Yeh et al., 2002, 2004; Freudenthal et al., 2005), an important body of evidence supports now the participation of NF-κB as a key regulator of gene transcription for long-term storage of information in the nervous system. Furthermore, the presence of NF-κB at the synapses and local activation during memory consolidation may directly influence this process through posttranslational modifications of other proteins (Salles et al., 2014). The results reviewed here support that the activity of this family of transcription factors is necessary in the hippocampus for the storage and the persistence of memory in different learning tasks, and both in consolidation and reconsolidation. One of the future directions in the research of the NF-κB role in memory should be the precise characterization of the NF-κB function at the synapse. Another direction for future work is to elucidate the effector genes that are regulated by this transcription factor and the specific role of their protein products. Among the target genes of NF-κB, brain derived neurotrophic factor (bdnf), zif268 and camk2d are candidates for future research efforts in the elucidation of the NF-κB-dependent gene regulation of key proteins that allow memory formation and particularly in the case of the latter gene, in the determination of more persistent memories.

#### Acknowledgments

We thank Dr. Liliana Orelli for helpful comments on the manuscript. The work reviewed here was supported by research grants from the National Agency of Scientific and Technological Promotion of Argentina (ANPCyT) PICT1482 and PICT2369, National Council of Research (CONICET) PIP5466 and University of Buenos Aires grant X198.

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**Conflict of Interest Statement**: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 de la Fuente, Federman, Zalcman, Salles, Freudenthal and Romano. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# NF-KappaB in Long-Term Memory and Structural Plasticity in the Adult Mammalian Brain

Barbara Kaltschmidt <sup>1</sup> \* and Christian Kaltschmidt <sup>2</sup>

*<sup>1</sup> Molecular Neurobiology, University of Bielefeld, Bielefeld, Germany, <sup>2</sup> Cell Biology, University of Bielefeld, Bielefeld, Germany*

The transcription factor nuclear factor kappaB (NF-κB) is a well-known regulator of inflammation, stress, and immune responses as well as cell survival. In the nervous system, NF-κB is one of the crucial components in the molecular switch that converts short- to long-term memory—a process that requires *de novo* gene expression. Here, the researches published on NF-κB and downstream target genes in mammals will be reviewed, which are necessary for structural plasticity and long-term memory, both under normal and pathological conditions in the brain. Genetic evidence has revealed that NF-κB regulates neuroprotection, neuronal transmission, and long-term memory. In addition, after genetic ablation of all NF-κB subunits, a severe defect in hippocampal adult neurogenesis was observed during aging. Proliferation of neural precursors is increased; however, axon outgrowth, synaptogenesis, and tissue homeostasis of the dentate gyrus are hampered. In this process, the NF-κB target gene PKAcat and other downstream target genes such as Igf2 are critically involved. Therefore, NF-κB activity seems to be crucial in regulating structural plasticity and replenishment of granule cells within the hippocampus throughout the life. In addition to the function of NF-κB in neurons, we will discuss on a neuroinflammatory role of the transcription factor in glia. Finally, a model for NF-κB homeostasis on the molecular level is presented, in order to explain seemingly the contradictory, the friend or foe, role of NF-κB in the nervous system.

#### Edited by:

*Benedict C. Albensi, University of Manitoba, Canada*

#### Reviewed by:

*Arturo Gabriel Romano, University of Buenos Aires, Argentina Subhajit Dasgupta, Saint James School of Medicine, Anguilla*

#### \*Correspondence:

*Barbara Kaltschmidt<EMAIL_ADDRESS>
> Received: *01 June 2015* Accepted: *30 October 2015* Published: *24 November 2015*

#### Citation:

*Kaltschmidt B and Kaltschmidt C (2015) NF-KappaB in Long-Term Memory and Structural Plasticity in the Adult Mammalian Brain. Front. Mol. Neurosci. 8:69. doi: 10.3389/fnmol.2015.00069* Keywords: NF-kappaB, long-term memory, transcription factors, synapse, dentate gyrus, long term potentiation

# WHAT IS MEMORY?

In its simplest form, we can define memory as information that can be retrieved later when required. The frequently used definitions of memory in psychology are called declarative memory and non-declarative memory. These terms can be used to discriminate memories of tasks such as riding a bicycle (non-declarative) from other tasks such as remembering a lecture, events, or places (declarative). As one might see with this simple example, there is no easy way to describe how information from the real world (outer world) is stored within the brain. Current knowledge describes what is necessary and sufficient for long-term memory formation such as neurons, action potentials, synapses, engram cells, and neuronal networks. The term 'engram' was coined by Richard Wolfgang Semon, who was the first to call memory traces as engram (Semon, 1920). There are several hypotheses involving single synapses, such as the synaptic tagging and capture hypothesis (Redondo and Morris, 2011), or involving engram cells and their plasticity in neuronal networks (Ryan et al., 2015 and see below). It seems to be unclear that which information of the real world is stored and how so many different memories acquired during lifetime can be stored and retrieved. A standard approach to memory research is that the observation, i.e., the information from the real world, is taken up by sensory organs, e.g., eyes, and then the information is transformed into signals, which are transmitted to other neurons in the form of ion flux, generating action potentials (neuronal activity). Long lasting storage of this neuronal activity is thought to be stored in small subnets of neurons within the brain. These neurons fire together, show correlated responses, and have strong synaptic connections, which may be used for memory storage (see Mayford et al., 2012 for review), whereas most synapses, not integrated in the investigated subnet, have only weak connections. Kandel and coworkers provided another definition for memory: "Learning is the biological process of acquiring new knowledge about the world, and memory is the process of retaining and reconstructing that knowledge over time" (Kandel et al., 2014), suggesting that memory retrieval could alter memory itself. For additional details on electrophysiology and memory, see Section Target Genes with Potential Involvement in Memory.

Pioneering work of Eric Kandel and coworkers has led to the identification of molecular pathways that translate changes of gene expression to behavior. This process involves intracellular protein phosphorylation and later on the nuclear transcription factor CREB as a target of the protein kinase A (Kandel, 2001). These findings inspired the interest of investigating transcription factors as regulators of memory traces, taking up an old concept from Richard Semon, who called memory traces engram (Semon, 1920): "Ich bezeichne diese Wirkung der Reize als ihre engraphische Wirkung, weil sie sich in die organische Substanz sozusagen eingräbt oder einschreibt. Die so bewirkte Veränderung der organsichen Substanz bezeichne ich als das Engramm des betreffenden Reizes. . . " Own translation to English: "I call this action of a stimulus an engraphic action, because it engraves or writes into the organic substance. This change in the organic substance is called engram of the corresponding stimulus." Recently Susumo Tonegawa and coworkers identified engram cells that retain memory even under retrograde amnesia (Ryan et al., 2015). They labeled engram cells by transcription of the activity-dependent c-fos promoter, driving tTA-dependent expression of either mCherry or chanelrhodopsin. Engram cells showed vast changes in synaptic plasticity on many spines. This could be fully inhibited by inhibiting protein synthesis. For an in depth discussion of the engram concept, see Ramirez et al. (2014). This work underscores the importance of transcription factors such as c-FOS for the formation of long-term memory in specific neurons (engram cells). We conclude that long-term memory could be stored in specific neurons (engram cells) and that engram cells are tagged during memory acquisition by the activation of transcription factors.

# TRANSCRIPTION FACTORS AND MEMORY

As discussed previously, transcription factors might provide a way to measure memory traces in neuronal networks, which are involved in memory storage. What is the definition of a transcription factor? A recent census of human transcription factors was done by identifying proteins that bind DNA in a sequence-specific manner (Vaquerizas et al., 2009). For this analysis, DNA-binding domains and families from the Interpro database were assembled and used for database search. This led to the identification of 1391 transcription factors in the human genome. However, only 62 of these putative transcription factors have been experimentally verified for both DNA-binding and regulatory functions. The authors reported among the most-cited transcription factors such as p53, FOS, JUN, CREB, and NFkappaB. Our own PubMed search in 2015 with MeSH entries "p53 and brain" resulted in 721 publications, whereas inclusion of "memory" in this search resulted in only 29 publications. Another PubMed search with "FOS and brain" resulted in 1399 publications, and with the additional search term "memory," 76 publications were found. About 3196 publications were found for "CREB and brain"; 796 hits were found with the addition of search term "memory." These results might underscore the importance of publications by Noble Prize winner Eric Kandel, who advanced the work on transcription and memory, especially on CREB (Kandel, 2001). Still NF-κB with MeSH term "brain" gives 1430 publications, and together with "memory," we received 108 publications. Reading of the abstracts immediately revealed that some important work was not found by these search strategies. Therefore, here, we will review the current literature on NF-κB and memory in mammalian systems. Besides NF-κB, other transcription factors activated by neuronal activity and involved in memory include cAMP response element-binding protein (CREB), CCAAT enhancer-binding protein (C/EBP), activating protein 1 (AP-1), and early growth response factor (Egr) (Alberini, 2009). Certainly there could be more.

# INTRODUCTION TO NF-κB

NF-κB is a master transcription factor that is ubiquitously expressed and responds to diverse stimuli including cytokines, growth factors, and bacteria or viruses by the expression of stress response genes in many cells (Hayden and Ghosh, 2012). NF-κB (nuclear factor kappa B) was discovered in the laboratory of David Baltimore (Sen and Baltimore, 1986), as a DNA-binding factor that is specific to a 10-base pair nearly palindromic sequence: 5′ -GGGACTTTCC-3, initially binding to the enhancer of the antibody kappa-light chain. Hence, this might explain its name. Cloning revealed a heterodimer composed of p50 kDa and p65 kDa (RelA) subunits. Further research identified other transcription factors with homology to the reticuloendotheliosis virus of turkeys (v-Rel). Each family member contains a Rel Homology region (RHR) near its N-terminus (**Figure 1**). The RHR contains two domains, the N-terminal domain (NTD) and the Dimerization domain (Dim), joined by flexible linker sequences. In addition, RHR contains a nuclear localization signal (NLS). Therefore, the RHR combines sequence-specific DNA-binding, dimerization, nuclear localization, and interaction with IκB proteins. IκB was purified by Patrick Baeuerle as a 60 to 70-kD inhibitory protein (called I kappa B) from a latent form of NF-κB in the cytoplasm, where latent NF-κB could be activated by treatment with mild detergent deoxycholate

can exist in a latent form in the cytoplasm complexed by IκB family members. This leads to inhibition of NF-κB due to cytoplasmic retention. Furthermore, NF-κB subunits can be classified as Class I (without transactivation, TA) or as Class II with TA due to the presence of a transactivation domain (TAD) in the C-terminal region.

(Baeuerle and Baltimore, 1988a). These data suggested a noncovalent interaction of IκB with NF-κB, which could be purified and cloned (Baeuerle and Baltimore, 1988b). Later, many different proteins with IκB function were identified (Hinz et al., 2012).

# ACTIVATION OF CREB AND NF-κB IN NEURONS

NF-κB is a transcription factor composed of two DNA-binding subunits that is activated by various stimuli in the nervous system (see **Figure 2**; Kaltschmidt and Kaltschmidt, 2009). Therefore, knockout or genetic ablation has various effects within the nervous system. In contrast, only knockout of subunit RelA (p65) is embryonically lethal. Genetic evidence for a role of NFκB in the nervous system including cognitive effects or other neurological defects are summarized in **Table 1**.

A comparison of NF-κB in the nervous system and CREB activation is shown in **Figure 2**. The CREB family contains CREB (cyclic-AMP-response element (CRE)-binding protein), CREM (CRE-modulatory protein), and ATF1 (activation transcription factor 1). Double-knockout of CREB and CREM resulted in a massive apoptotic cell death of neurons of the cortical plate and in the hippocampus and striatum, whereas neurogenesis seemed to be unaffected (Mantamadiotis et al., 2002), and no fiber defects were reported. Phosphorylation by protein kinase A at Ser133 activates CREB to promote transcription (for review, see West et al., 2002). Phosphorylation can lead to the recruitment of the transcriptional coactivator CREB-binding protein (CBP). CBP belongs to a family of histone acetyl-transferases, which catalyze hyper-acetylation of core histones, thereby leading to gene activation by loosening chromatin packing. Neuromodulators such as dopamine can activate G-protein-coupled receptors,

FIGURE 2 | Schematic representation for the activation of transcription factors like NF-κB and CREB by synaptic activity. Upon synaptic stimulation of the receptors for NMDA or voltage-gated calcium channels (L-VGCC), a calcium influx induces activation of IkB kinases and phosphorylation of IkB (blue). Finally, degradation within the proteasome takes place, and thereby the nuclear localization signal of NF-kB is unmasked and allows NF-κB (red/green) to enter the nucleus, where it binds to kb sites and activates the transcription of target genes like PKAcat (catalytic subunit of protein kinase A). On the left site, CREB is activated after stimulation of mGluR or dopamine receptors by dopamine, and cAMP increases activating PKA. This phosphorylates the transcription factor CREB and other proteins like MAP kinases. P-CREB (orange) is bound by CBP (yellow), which initiates the transcription of its target genes. Interestingly, activation of NF-κB and expression of its target gene PKAcat will induce a positive feed-forward loop to CREB activation without additional synaptic activation, which might explain the late phase of long-term potentiation.

which in turn activates membrane localized adenylate cyclase, which leads to the elevation of intracellular cAMP (**Figure 2**). Activation by cAMP frees the catalytically active protein kinase A (PKAcat) to enter the nucleus and phosphorylate CREB, which could ameliorate age-dependent memory deficits (Bach et al., 1999). Neuromodulators binding to metabotropic glutamate receptors can also activate NF-κB in neurons (O'Riordan et al., 2006). Activation of plasma membrane-localized ion channels, such as NMDA (N-methyl-D-aspartate) receptors (NMDARs) and L-type voltage-gated calcium channels (L-VGCCs), allow the influx of calcium (**Figure 2**). The classical view was that CREB could be activated by phosphorylation via the depolarizationactivated Ca(2+) -calmodulin-dependent protein kinases (CaM kinases) I and II (Sheng et al., 1991). Then, how the calcium signal is transmitted to the nucleus? This story was unfolded about 25 years later. Recent results suggest a CaM kinase cascade (see **Figure 2**), where the calcium signal is transmitted via calmodulin-dependent phosphorylation of gamma CaMKII. The activated γCaMKII travels to the nucleus where phosphorylation of CREB is initiated (Ma et al., 2014). Until now, there are only two transcription factors known to decode frequency of calcium oscillations: NFAT and NF-κB (Dolmetsch et al., 1998). While NF-κB is already activated by low frequency of calcium oscillations and by infrequent oscillations even at the level of its target genes IL2 and IL8 (Dolmetsch et al., 1998), nothing

system.

**19**

is known about the situation in neurons. It is suggested that NF-κB could be activated by calcium in cerebellar granule cells (Lilienbaum and Israël, 2003). Further investigations might clear the mechanisms in other neuronal cell types. All NF-κB-

Kaltschmidt and Kaltschmidt NF-KappaB and Memory neurons with tumor necrosis factor alpha (TNF) or low amounts of amyloid beta peptide (Aβ) could protect neurons against Aβ-mediated neurotoxicity (Kaltschmidt et al., 1999). In this line, it could be shown that TNF is involved in hippocampal synaptic plasticity (Albensi and Mattson, 2000). Furthermore, we have previously shown (Kaltschmidt et al., 1999) that treatment of cerebellar granule cells with TNF or low dose Aβ peptide (0.1µM) could protect against neurotoxic amounts of Aβ (10µM). This type of neuroprotection involves NF-κB and might be enhanced by CBP as a potential NF-κB target gene. CBP is a co-activator, which seems to be important for chromatin opening, due to its histone acetyltransferase activity (Ogryzko et al., 1996). Interestingly, several neuroprotective small molecules could be identified by a screening strategy using

activating stimuli culminate on the IKK complex composed from the IκB kinases IKK 1 and 2 (alpha and beta) and the third subunit NEMO (Israël, 2010). Activation of the IKK complex could involve ubiquitinylation of NEMO and upstream kinases, and the entire mechanism is not solved so far. Activation of the IKK complex leads to phosphorylation of the inhibitory subunit IκB alpha at Ser 32 and 36 followed by ubiquitinylation. This is a signal for proteolytic degradation of IκB in the proteasome. Anti-cancer drugs such as bortecomib (Velclade) are used to inhibit constitutive NF-κB activity in multiple myeloma plasmacytoma cells. In the context reviewed here, bortecimib can have serious side effects on the nervous system (see Velcade EMA/27714/2015) such as posterior reversible encephalopathy syndrome, autonomic neuropathy (damage to nerves controlling organs such as the bladder, eyes, gut, heart, and blood vessels), or more commonly peripheral neuropathy (nerve damage in hands and feet). Furthermore, inhibition of the proteasome system during consolidation blocked long-term memory both in crabs and mice (Sol Fustiñana et al., 2014). Degradation of IκB frees DNA-binding subunits to enter the nucleus by active transport (**Figure 2**), leading to the expression of target genes such as PRKACA (catalytic subunit of PKA; Kaltschmidt et al., 2006). NF-κB could be activated in neurons by depolarization, kainate, and glutamate (Kaltschmidt et al., 1995; Simpson and Morris, 1999). Stimulation in young cerebellar granule cells functions even at the concentration of 10 nM glutamate, alone or together with 10µM glycine or 100µM NMDA (Guerrini et al., 1995); interestingly, neurons prepared from older age depicted constitutive NF-κB activity as already reported for hippocampal and cortical neurons in vitro and in vivo (Kaltschmidt et al., 1994). This constitutive NF-κB activity could be modulated by co-cultured astrocytes, which could inhibit activation of NFκB in vitro (Kaltschmidt and Kaltschmidt, 2000). Constitutive NF-κB activity is also found in vivo when reporter genes were analyzed.  Expression of reporter genes was only neuronal. Furthermore, forced expression of transdominant IκB alpha completely blocked constitutive expression of this lacZ NFκB reporter gene (Fridmacher et al., 2003). Similar results NF-κB activation (Manuvakhova et al., 2011). Memory decline is one of the hallmarks of Alzheimer's disease, which could be correlated to reduced NF-κB activity around Aβ containing plaques (Kaltschmidt et al., 1999). Neuroprotection against kainate was blocked by forced expression of transdominant IκB remains.

were achieved with another mouse model (Bhakar et al., 2002) using a κB tandem repeat derived from HIV LTR to drive an SV40 minimal promoter lacZ gene. LacZ expression was strong in the telencephalon and along the roof plate of midbrain. Among other loci of staining, tactile hair follicles were prominently stained. Layers of the cortex cerebri are stained to a lesser extent, but strong staining was evident in hippocampal fields CA1, CA2, DG, and to a lesser extent in CA3. Forced expression of RelA protected cortical neurons against apoptosis induced by etoposide (Bhakar et al., 2002). We found that pre-conditioning alpha (Fridmacher et al., 2003). In **Figure 2**, as an example of a target gene, the catalytic subunit of protein kinase A (PKAcat) is depicted; this gene is regulated by NF-κB in hippocampal neurons in vivo (Kaltschmidt et al., 2006). Forskolin-induced (cAMP-dependent) CREB phosphorylation is strongly impaired when NF-κB is repressed. These data suggest a cross-coupling of CREB and NF-κB-dependent memory pathways. However, the question which are the relevant memory target genes still

# TARGET GENES WITH POTENTIAL INVOLVEMENT IN MEMORY

Long-lasting long-term potentiation (L-LTP) is a well-established model for memory at the synaptic level. In this paradigm, short voltage trains are used to induce a long-lasting post-synaptic response. It was discovered by Lømo when stimulating the perforant path (input to dentate gyrus granule cells) (Bliss and Lømo, 1973). Recording of field potentials of granule cells showed a long-lasting potentiation of granule cells lasting for hours (L-LTP). The requirement of L-LTP for protein synthesis was first described by Krug et al. (1984). In vivo L-LTP resulted in long-lasting biphasic CREB phosphorylation (Schulz et al., 1999). NF-κB p50−/<sup>−</sup> animals showed only decremental LTP that faded over time, even when high frequency stimulation trains were used, which elicited L-LTP in control animals (Oikawa et al., 2012). Furthermore, LTP magnitude and LTD induction were blocked by inhibiting NF-κB DNA binding in hippocampal slices (Albensi and Mattson, 2000). In this line, expression of Egr-2 was upregulated in WT hippocampal slices preparation after theta burst stimulation but not in p50−/<sup>−</sup> slices (Nafez et al., 2015). Same authors identified Egr-2 as novel NF-κB target gene in brain. Knockout of c-Rel also led to decremental LTP, whereas c-Rel expression was up-regulated during novel object recognition (Ahn et al., 2008). Late-onset Parkinson's disease was reported in mice with c-Rel knockout with loss of tyrosine hydroxylase positive neurons in the substantia nigra only visible at 18 month of age (Baiguera et al., 2012). Inhibition of all NF-κB subunits by forced IκB expression led to reduced LTP induction and longer learning phases in Morris water maze (Kaltschmidt et al., 2006). As potential mechanisms, the current models discuss the formation of novel post-synapses and NMDA receptor density increase. Previous reviews suggested that the two secreted proteins, brain-derived neurotrophic factor (BDNF) and tissue plasminogen activator (tPA), have been repeatedly implicated in L-LTP as memory-relevant CREB target genes (Kandel, 2001) or nNOS and presenilin as additional CREB targets (West et al., 2002). To analyze this question in more detail, Eric Kandel and coworkers expressed a constitutively transactivating mutant of CREB (VP16-CREB) in transgenic mice under the control of the tetracycline operator, which can be activated by CamKII promoterdriven expression of the tetracycline-dependent transactivator (Barco et al., 2005). Surprisingly, this study identified only a handful of genes that are activated by VP16-CREB-induced L-LTP, but not by increased neuronal activity alone (kainate treatment). These genes include prodynorphin, MHC Class I and JunD/cFos in CA1, and dentate gyrus (see **Figure 3**). A low amount of induction in all hippocampal subfields was detected for BDNF. Surprisingly, Arc was only induced by kainate treatment, suggesting a difference in genes activated by neuronal activity and memory-relevant gene expression. Furthermore, MHC Class II genes seemed to be induced by VP16-CREB. These data could be reproduced in part with a cellular model of cultured hippocampal neurons (Benito et al., 2011). One might be surprised by the fact that a similar set of genes were identified in a different but technically similar in vivo screen, involving loss of function of NF-κB (Kaltschmidt et al., 2006). Therefore, we suggest that JunD, MHC Class I, II and beta 2 microglobulin, prodynorphin, and BDNF might be a common set of genes that are regulated by CREB and NF-κB together. Indeed, MHC Class I, II and beta 2 microglobulin share common

response elements including the CREB-binding site, CRE (Gobin et al., 2001). Similarly, these genes are NF-κB target genes (Israël et al., 1989) even in neurons (Kaltschmidt et al., 1995; Yang et al., 2014). However, function in neurons remained unclear. MHC Class I molecules are trimeric proteins that are composed of a transmembrane heavy chain, a soluble κ2-microglobulin (κ2 m) light chain, and a peptide bound to the heavy chain. Only recently, Carla Shatz and coworkers have suggested a synaptic glue hypothesis (Huh et al., 2000), where MHC Class I molecules might be used for adhesion of specific synapses. Surprisingly, LTP is enhanced and LTD is absent in mice without functional MHC Class I (Huh et al., 2000). Expression of MHC Class I genes H2-D and T22 could be detected in the hippocampus (Huh et al., 2000). However, other authors argue that MHC Class I has a function in pathological situations (e.g., synapses and neuron elimination by T-cells; Cebrián et al., 2014). Similarly, no memory-relevant target genes for the AP-1 complex (Fos/Jun) were presented so far. Prodynorphin was identified as a NF-κB target gene with mapped response sites (Bakalkin et al., 1994). BDNF expression might be regulated indirectly via the NF-κB target gene XIAP (Kairisalo et al., 2009). In our humble opinion, the correlation with memory formation is better for NF-κB target genes (**Figure 3** right part of the Venn diagram). The initial idea of synaptic enhancement by transcription is of a target gene that can indeed enhance post- or pre-synapse function. We think there are now, after more than 20 years of research on NFκB in the nervous system, enough evidences on involvement of NF-κB target genes in synaptic enhancement. In this line, a recent genetic screen identified an insulin-like growth factor 2 (Igf2, see **Figure 4**) as a novel neuronal NF-κB target gene involved in spine density regulation (Schmeisser et al., 2012). Interestingly, Igf2 could rescue the spine reduction in neurons with NF-κB inhibition (Schmeisser et al., 2012). In addition, Igf2 is critically involved in memory consolidation and enhancement (Chen et al., 2011). Igf2 could ameliorate the neurodegeneration in a mouse

model with Alzheimer's disease, and its expression is reduced in Alzheimer patients' hippocampi (Pascual-Lucas et al., 2014). Similarly, NF-κB activity was reduced in Alzheimer patients' brains (Kaltschmidt et al., 1999), suggesting that Igf2 could be a target gene of NF-κB in humans. In addition, amyloid beta peptides could activate NF-κB p65 in neurons and might activate NF-κB in the close vicinity of early plaques in Alzheimer patients' brains (Kaltschmidt et al., 1997). In this respect, it might be important to note that the activation of NF-κB by Alzheimer's disease follows a bell-shaped curve where low amounts of Aβ activate NF-κB, but high amounts repress NF-κB; for discussion, see Kaltschmidt et al. (2005). In a recent study (Schmeisser et al., 2012), a dominant negative version of the IKK2 protein was expressed in neurons using the tet system. Spine density was reduced to the half of control in CA1, especially the memoryrelevant mushroom spines were reduced to about 25%. Learning was slower as with IκB over-expression. Synapses contained significantly less GluA1 and PSD95. Ultrastructure depicted a high amount of synapses with disrupted post-synaptic density. Similarly, it was shown earlier in cultured neurons that NF-κB activity positively regulated spine densitiy and glutamatergic synapse formation (Boersma et al., 2011). These data describing post-synaptic damage fit to our data reporting reduced size and number of presynaptic mossy fiber buttons (Imielski et al., 2012). In addition, we observed that axonal growth is regulated by NF-κB in vitro and in vivo in the mossy fiber pathway (Imielski et al., 2012). Taken together, NF-κB regulates postsynapse formation (see **Figure 4**) by expression of Igf2, whose receptor is post-synaptic and by additional target genes such as PSD95 (Boersma et al., 2011). Furthermore, NF-κB directs the expression of AMPA receptor components (Boersma et al., 2011; Schmeisser et al., 2012). NMDA receptor component NR1 appears to be regulated by NF-κB, and regulation of NF-κB target genes such as Cox2 (Kaltschmidt et al., 2002), PKAcat (Kaltschmidt et al., 2006), Foxo1, and NCAM (Imielski et al., 2012) were described. Localization of NF-κB in synaptosomes (Kaltschmidt et al., 1993; Meberg et al., 1996; Meffert et al., 2003; Schmeisser et al., 2012) suggests a function of NF-κB as retrograde messenger (see **Figure 5**). In this line, we and others could show that p65-GFP is traveling to the nucleus when activated by glutamate (Wellmann et al., 2001; Meffert et al., 2003). This retrograde transport is dependent on the nuclear localization signal of p65 and involves a dynein–dynactin motor protein complex traveling on microtubules (Mikenberg et al., 2007; Shrum et al., 2009). The traveling complex seems to be a signaling endosome with scaffolding proteins such as Huntingtin and/or HSC70 (Marcora and Kennedy, 2010; Klenke et al., 2013). In comparison, it might be interesting to note that CREB could be translated in axons and mediate a retrograde survival signaling in response to nerve growth factor (Cox et al., 2008).

# NF-κB SIGNALING IS ESSENTIAL FOR DENTATE GYRUS TISSUE HOMEOSTASIS

Previously, we have shown that NF-κB plays an important role at three consecutive stages of neurogenesis (see **Figure 6**):

proliferation/differentiation of neural progenitor cells, axon specification, and integration of young neurons and survival of mature granule cells (Imielski et al., 2012). Recently, the role of NF-κB p50 in neurogenesis was analyzed (Denis-Donini et al., 2008) in p50−/<sup>−</sup> mice, where the net rate of neural precursor proliferation was unchanged, but only 50% of newborn neurons survived in the DG and a defect in spatial shortterm memory was observed. Because in p50−/<sup>−</sup> mice only one NF-κB subunit is deleted in all cell types, we used (Imielski et al., 2012) a neuronal-specific ablation of all NF-κB subunits in our study. We confirmed in part the results of Denis-Donini and coworkers for a role of NF-κB in neurogenesis and extended that findings to a function of NF-κB in tissue of the dentate gyrus homeostasis, synaptogenesis, axogenesis, and spatial pattern separation. In contrast to Denis-Donini et al., we found an increased rate of proliferating DCXpositive granule cell progenitors, presumably due to the high rate of apoptosis observed in DG after forced expression of transdominant negative IκB-alpha. Regrowing of DG after reactivation of NF-κB had a major impact on integration and survival of newborn DCX+ neurons. Similarly, neurogenesis within the subventricular zone depends on NF-κB (Zhang et al., 2012).

The regulation of adult neurogenesis by transcription factors is still a matter of debate (Chen et al., 2011), and our data show that the transcription factor NF-κB is a crucial regulator of neurogenesis, essential for axogenesis, and integration of newborn neurons. Taken together, these data indicate that NF-κB plays an important role in structural plasticity of the hippocampus.

Surprisingly, another study showed that over-activation of NF-κB in a transgenic mouse model with forced expression of ncaIKK-2 (Maqbool et al., 2013) resulted in a similar structural defect: degeneration of granule cells to about 50% of control level and massive astrocytosis. In addition, a reduction of BDNF was described but no decrease of Prkca was measured. However,

expression of the NF-κB target gene Igf2 was not elevated in this mouse model, arguing against the validity of gain of function approaches to study NF-κB target genes.

To the best of our knowledge, NF-κB is the one and only factor where ablation or over-activation showed such severe effects in structural plasticity.

The observed NF-κB-dependent structural defects resulted in a behavioral phenotype. Recently, behavioral tests (Clelland et al., 2009) were developed to measure the memory of subtle differences in spatial environment. The authors suggest that neurogenesis enhanced the recognition of weakly separated cues. In this line, we developed a special behavioral test (SPS-BM), which is able to measure spatial pattern separation with the advantage to analyze search strategies (Widera et al., 2014).

Interestingly, the mouse model described in Imielski et al. (2012) is a phenocopy of Alzheimer's disease (AD) in three aspects: reduced NF-κB activity as in AD brains (Kaltschmidt et al., 1999), increased proliferation of immature DCX+ neuronal precursors, and progressive cell loss (Jin et al., 2004) coupled with strong neuroinflammation. Thus, re-activation of NF-κB might

#### REFERENCES

be an interesting therapeutic strategy for neuroregeneration of the adult dentate gyrus in the future.

# CONCLUSION

We suggest that NF-κB activation in neurons could be an interesting strategy to ameliorate memory diseases such as Alzheimer's disease. Dose–response curve of such kind of drugs might be bell shaped, too much will lead to neurodegeneration. Furthermore, constitutive activation of NF-κB in glia during aging might lead to cognitive decline. Finally, re-activation of NF-κB inhibition might lead to neuroregeneration due to stem cell action, which could be the basis for further understanding of brain repair.

#### ACKNOWLEDGMENTS

We thank the German Research Council (DFG), the Walcker Stiftung, Isny, Germany, and the University of Bielefeld for funding our research.

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peripheral myelin formation. Nat. Neurosci. 6, 161–167. doi: 10.1038/ nn995

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Kaltschmidt and Kaltschmidt. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# A new perspective on the role of the CREB family of transcription factors in memory consolidation via adult hippocampal neurogenesis

#### Sylvia Ortega-Martínez\*

Turku Centre for Biotechnology, Åbo Akademi University and University of Turku, Turku, Finland

Adult neurogenesis is the process by which new neurons are generated in the brains of adults. Since its discovery 50 years ago, adult neurogenesis has been widely studied in the mammalian brain and has provided a new perspective on the pathophysiology of many psychiatric and neurodegenerative disorders, some of which affect memory. In this regard, adult hippocampal neurogenesis (AHN), which occurs in the subgranular zone (SGZ) of the dentate gyrus (DG), has been suggested to play a role in the formation and consolidation of new memories. This process involves many transcription factors, of which cyclic AMP (cAMP)-responsive element-binding protein (CREB) is a well-documented one. In the developing brain, CREB regulates crucial cell stages (e.g., proliferation, differentiation, and survival), and in the adult brain, it participates in neuronal plasticity, learning, and memory. In addition, new evidence supports the hypothesis that CREB may also participate in learning and memory through its involvement in AHN. This review examines the CREB family of transcription factors, including the different members and known signaling pathways. It highlights the role of CREB as a modulator of AHN, which could underlie its function in memory consolidation mechanisms.

Keywords: adults, mammals, cyclic AMP-responsive element-binding protein, hippocampus, memory, neurogenesis, neuronal plasticity, transcription factors

# The CREB Family of Transcription Factors

Cyclic AMP (cAMP)-responsive element-binding protein (CREB) belongs to the family of leucine zipper transcription factors, which are expressed in a variety of tissues (Yamashima, 2012). In 1986, Marc Montminy and R.H. Goodman first defined cAMP response element (CRE) as a conserved DNA sequence in promoter elements that were activated by cAMP. One or several copies of CRE are present within the regulatory regions of genes. Binding of different transcription factors to CRE regulates RNA polymerase activity, thereby controlling gene expression. A year after CREB was defined, Montminy and Bilezikjian (1987) described CREB as a cellular transcription factor that binds CRE and induces transcription of the somatostatin gene.

The CREB family of transcription factors is now known to include CREB, CRE modulator (CREM), and activating transcription factor-1 (ATF-1). This family belongs to the superfamily of

#### Edited by:

Benedict C. Albensi, University of Manitoba, Canada

#### Reviewed by:

John Oberdick, The Ohio State University, USA Baojin Ding, University of Massachusetts Medical School, USA

#### \*Correspondence:

Sylvia Ortega-Martínez, Turku Centre for Biotechnology, Åbo Akademi University and University of Turku, Tykistokatu 6, FI-20520 Turku, Finland<EMAIL_ADDRESS>
> Received: 16 February 2015 Accepted: 11 August 2015 Published: 26 August 2015

#### Citation:

Ortega-Martínez S (2015) A new perspective on the role of the CREB family of transcription factors in memory consolidation via adult hippocampal neurogenesis. Front. Mol. Neurosci. 8:46. doi: 10.3389/fnmol.2015.00046

**Abbreviations:** CREB, cAMP-responsive element-binding protein; AHN, adult hippocampal neurogenesis; DG, dentate gyrus; SGZ, subgranular zone; SVZ, subventricular zone; KID, central kinase-inducible domain.

basic region/leucine zipper (bZIP) transcriptional regulators, and all its members mediate cAMP-responsive transcription. CREB and ATF-1 are expressed ubiquitously, whereas CREM is mainly expressed in the neuroendocrine system. Each transcription factor is encoded by a single gene and contains the highly conserved leucine zipper sequence with an adjacent basic region that binds to a CRE DNA sequence. Members of the CREB family also have a regulatory central kinase-inducible domain (KID), although the Q1 and Q2 glutamine-rich regions adjacent to KID are less conserved among different family members (see review Barco et al., 2003).

Creb1 is the gene responsible for transcription of all known CREB isoforms; the α isoform is the most common, but the β and ∆ isoforms are also transcribed. Several approaches have been used to clarify the roles of different CREB isoforms. One study assessed CRE-DNA binding in nuclear extracts obtained from several brain regions of wild-type and CREB∆/α mutant mice. The results suggested that CRE-DNA–binding complexes contain both CREB and CREM proteins. However, CRE-DNA binding was abolished in the cortex, hippocampus, cerebellum, and amygdala of CREB∆/α mutant mice. Because these transgenic animals lack CREB∆ and α isoforms, the expression of other forms of CREB, such as CREB-β and CREM, is upregulated. The findings indicate that CREM binding to CRE sites requires the presence of CREB∆/α, and that CREB-β may not efficiently bind to CRE sites (Pandey et al., 2000). In addition, mice lacking the α and ∆ CREB proteins have abnormal long-term, but not short-term memory (STM), indicating that these isoforms are essential for the role of CREB in long-term memory (LTM; Bourtchuladze et al., 1994). In contrast to previous classical studies in adult mice, some studies have focused on the effect of these isoforms on neocortical plasticity in young mice to highlight the agedependent role of CREB in neuroplasticity. In one of these studies, the CREBα/∆ mutation did not affect plasticity in cortical layers II/III of the younger adolescent mice (1–2 months) indicating that different plasticity processes occur at this age. In the same model, CREBβ expression was upregulated in the barrel cortex of CREBα/∆ knock-out animals, suggesting that this subunit may partly compensate for the loss of the α/∆ isoforms in the young mice. Overall, the study results suggested that CREB isoforms play a role in experiencedependent plasticity in the adult neocortex (Glazewski et al., 1999).

The Creb1 gene has a complex structure with multiple exons and introns that result in several alternatively spliced mRNAs encoding proteins with distinct transcription-activating or transcription-repressing properties. However, two structural features are present in most members of the CREB family: (a) the well-conserved bZIP domain at the C-terminus, which facilitates dimerization between different family members and participates in the recognition of and binding to CRE sites; and (b) KID (mentioned above), which encloses sites for phosphorylation by protein kinase A (PKA) and other kinases. KID is delineated by two glutamine-rich domains (Q1 and Q2), which are responsible for basal transactivation activity. Q2 connects with TAFII 130 (TBP [TATA-binding protein]-associated factorII 130) and recruits the transcription machinery to the promoter region. Constitutive (Q2) and inducible (KID) domains work together in response to different stimuli that trigger CREB-dependent gene expression (see review Barco et al., 2003). Transcription factors of the CREB family exert their actions as homodimers or heterodimers, but the CREB-CREB homodimer is the most potent transcriptional activator (Dworkin and Mantamadiotis, 2010). CREB isoforms are widely distributed in the adult mouse brain under homeostatic conditions and remain inactive when bound to CRE elements within target gene promoters (Nichols et al., 1992).

The transcriptional activity of CREB depends on its phosphorylation status, which is determined by the opposing actions of protein kinases and phosphatases. Phosphorylation is a key mechanism in signaling and has been described to regulate several processes such as the cell cycle (Liu et al., 2014), cell death (Martin, 2010), DNA damage (Abreu et al., 2013) and neurogenesis (Faigle and Song, 2013). For example, increase in intracellular calcium (Ca2+) levels through voltageand ligand-dependent channels results in increased cAMP levels via activation of G-protein-coupled receptors. Growth factors can also activate receptor tyrosine kinases to augment cAMP levels. All these pathways affect CREB phosphorylation levels. Downstream of neuronal activity, the PKA, mitogen-activated protein kinase (RSK [p90 ribosomal protein S6 kinase]/MAPK), and CaMKIV (Ca2+/calmodulin-dependent protein kinase IV) kinase pathways result in CREB phosphorylation at Ser133. PP-1 (protein phosphatase-1) and PP2-A are the major CREB phosphatases, and Ca2+-dependent signals can induce intracellular events that lead to phosphorylation or dephosphorylation. The effect of stimulation depends on the nature of the stimulus and its cellular context. For example, activation of N-methyl-D-aspartate (NMDA) receptors leads to CREB dephosphorylation in extrasynaptic neurons, while in synaptic sites, it leads to CREB phosphorylation and CREBdependent gene expression (Ghiani et al., 2007).

The phosphorylation of a serine residue (S133) in KID regulates CREB activity. This phosphorylation establishes an effector role for CREB, initiating the translation of extracellular stimuli into gene expression (Yamashima, 2012). Indeed, CREB phosphorylation facilitates signal transduction, and molecules responsible for its phosphorylation/dephosphorylation are, therefore, highly relevant as modulators of the target gene status. There are a number of signaling cascades upstream of CREB phosphorylation, such as the cAMP pathway (Montminy and Bilezikjian, 1987), and protein kinase pathways (e.g., PKA and CaMKII and IV; Yamashima, 2012). These cascades are activated by stress, inflammatory cytokine activity (MAPK or phosphoinositide 3-kinase (PI3)/Akt pathways; Yamashima, 2012), growth factor/receptor tyrosine kinase activity (Ras/Erk/RSK2 pathway; Ghosh et al., 1994), and phospholipase C (PLC)-PKC signaling (see **Figure 1**). All these cascades have been shown to induce CREB phosphorylation (Du and Montminy, 1998; Lonze and Ginty, 2002). For example, CREB phosphorylation is facilitated by membrane-bound NMDA receptors (Ghiani et al., 2007), the non-receptor tyrosine protein kinase

c-Src (Zhao et al., 2003a), fibroblast growth factor receptor 1 (FGFR1; Hu et al., 2004), and estrogen receptors (Sharma et al., 2007), all of which are part of distinct intracellular cascades. The molecules involved in the modulation of CREB phosphorylation include several neurotransmitters [e.g., dopamine, glutamate, serotonin, gamma-aminobutyric acid (GABA)], growth factors (e.g., insulin-like growth factor 1 IGF-1; vascular endothelial growth factor, VEGF), and neurotrophins (brain-derived neurotrophic factor, BDNF; Yamashima, 2012). A summary of CREB pathways is depicted in **Figure 1**.

CREB-mediated transcription requires CREB phosphorylation, but the latter is not a reliable predictor of target gene activation; additional regulatory processes are required for the engagement of transcriptional promoter elements. For example, clustering of CBP (CREB-binding protein) has recently been described as crucial, and the CBP paralog p300 enhances CREB functionality. CBP and p300 are both chromatin remodelers (histone acetyltransferases) involved in unwinding promoter chromatin and providing the RNA polymerase II (Pol II) transcription machinery access to promoter DNA (Chan and La Thangue, 2001). If CBP/p300 does not cluster at a gene promoter after CREB phosphorylation, the gene remains transcriptionally silent (Merz et al., 2011). Thus, many co-factors, including some that are directly affected by neuronal activity and growth factors, influence CREB-mediated regulation at the final stage (Merz et al., 2011). This allows CREB signaling to control expression of a variety of target genes.

CREB was first isolated from undifferentiated neuron-like PC12 cells (Montminy and Bilezikjian, 1987) and from the mouse brain (Yamamoto et al., 1988). Interest in CREB's role in the nervous system has increased in the last few decades, and it has been proposed to be a key regulator of several complex processes ranging from development to plasticity (Yamashima, 2012). For example, CREB has been postulated as a regulator of cell survival, proliferation, and differentiation in the developing brain, whereas its roles in the adult brain include learning, memory, and neuronal plasticity (Yamashima, 2012). It is a key molecule involved in LTM in the majority of species (Bourtchuladze et al., 1994; Yin et al., 1994).

CREB participation in memory processes has been widely analyzed using animal models ranging from nematodes to higher mammals. The use of different CREB-mutant mice has been one of the most important approaches in elucidating CREB's role in these memory processes (see review Kida, 2012). CREB has been implicated in the regulation of embryonic and adult neurogenesis (Dworkin et al., 2009). Both neurogenesis and memory, which have been shown to be highly related, constitute an important field of research (see **Figure 2**). It is widely accepted that adult neurogenesis is involved in memory consolidation and pattern separation, and impairment of these processes has been described in various diseases associated with memory loss, including Alzheimer's disease (AD; Taupin, 2009; Braun and Jessberger, 2014; Fitzsimons et al., 2014; Cameron and Glover, 2015).

# Current Understanding of CREB, Neurogenesis, and Memory

There were fewer scientific publications on CREB until the 1990s after which the number of publications on this topic have increased. This rise has been accompanied by an exponential increase in publications on learning and memory. Although memory and learning have been studied for a long time, only recent advances in molecular and cellular techniques have enabled researchers to investigate the mechanisms underlying these processes. These advances have permitted the study of complex events such as gene expression in greater detail. Considering that the DNA helix was discovered in 1953, transcription factors (e.g., CREB) are relatively new concepts. Moreover, ideas about neurogenesis have evolved since its discovery 50 years ago (**Figure 2**).

This review focuses on the most relevant and recent studies on CREB's role in memory regulation and its participation in neurogenic processes. CREB influences cognitive processes directly by affecting memory and indirectly by affecting adult hippocampal neurogenic capacity. CREB's affects memory consolidation through its regulation of adult hippocampal neurogenesis (AHN), which mainly occurs in the hippocampal subgranular zone (SGZ) of the dentate gyrus (DG). This mechanism provides a novel perspective on memory consolidation within the adult hippocampus.

# Adult Neurogenesis

The existence of ''adult neurogenesis'' in mammals was first confirmed 50 years ago (Altman and Das, 1965). Adult neurogenesis refers to the generation of new neurons in the adult brain. Despite its importance, this emergent concept remained obscure until neurogenesis was found to occur in the brain of adult humans (Eriksson et al., 1998). Neurogenesis was traditionally understood to be mainly an embryogenetic phenomenon, but new research has shown the generation of new nerve cells in several areas of the adult brain, including the DG, subventricular zone (SVZ), olfactory bulb (OB), and other areas where it was recently observed (e.g., the cortex and hypothalamus (Gould, 2007). Adult neurogenesis involves cell proliferation, survival, and cell differentiation. AHN, which occurs for specific brain functions, involves not only new neuron formation but also integration of these new-born neurons into functional networks. From this perspective, AHN facilitates memory consolidation via formation of networks (Deng et al., 2010; Weisz and Argibay, 2012). Furthermore, AHN provides plasticity required in memory processes and allows for ''pattern separation mechanisms, '' which is crucial for memory consolidation (Bruel-Jungerman et al., 2007; Sahay et al., 2011a; Bekinschtein et al., 2013; Yassa and Reagh, 2013). It is critical to understand this phenomenon because neurogenesis also occurs after brain injury or during brain alteration (e.g., epilepsy); however, in these cases, it is a non-functional process as it is not related with their normal brain function and does not involve wiring of new neurons into networks (Zhang et al., 2014).

#### AHN and Memory

In the adult brain, neural stem cells (NSCs) are found in the SGZ, a specific DG subregion (Fanselow and Dong, 2010). These NSCs give rise to new neurons, which are incorporated as new granule cells into hippocampal networks. These networks contribute to learning (Zhao et al., 2003b; Joiner, 2009; Zhang et al., 2009) and memory (van Praag et al., 1999, 2002). In contrast, the ventral DG hippocampus (ventral SGZ) has been implicated in emotion-related disorders (Fanselow and Dong, 2010), including stress, depression, and anxiety. Studies on the types of memories related to AHN have highlighted its role in trace fear conditioning, recognition, spatial memory, pattern

separation, and non-mnemonic tasks (reviewed in Cameron and Glover, 2015).

Numerous studies have focused on the relationship between AHN and BDNF. It is widely accepted that BDNF stimulates neurogenesis (Zhang et al., 2011) and is expressed solely in the hippocampus with no additional sources from the peripheral blood. Increased BDNF levels have been shown to enhance hippocampal neurogenic capacity, resulting in improvements in hippocampus-dependent memory (Hsiao et al., 2014). The effects of voluntary exercise and enriched environments on BDNF levels and adult neurogenesis have also been widely studied (Bekinschtein et al., 2011; Yu et al., 2014). IGF-1, another growth factor involved in the BDNF pathway, was also found to affect AHN. Exercise increases IGF-1 levels, which leads to increments in AHN with subsequent improvements in memory and learning. Serotonin and noradrenaline release also upregulate BDNF, explaining the high BDNF levels and neurogenesis following antidepressant treatment (Albert and Benkelfat, 2013).

In summary, adult neurogenesis is considered essential for memory consolidation. Recently, neurogenesis has been discovered to occur in more brain areas; however, memoryrelated neurogenesis primarily occurs in the DG of the hippocampus and OB. The adult hippocampus has been widely studied for its role in the development of numerous neurodegenerative and neuropsychiatric disorders characterized by decreased AHN. Because hippocampus-dependent memory processes are altered in such diseases, understanding the underlying molecular mechanisms is important for restoring normal brain function. Indeed, some recent drug discovery efforts have focused on increasing AHN.

#### AHN and Olfaction

Neurogenesis attributed to the SVZ, the largest neurogenic niche, is also responsible for generating astrocytes, oligodendrocytes, and OB interneurons (Lois and Alvarez-Buylla, 1994); which are involved in ''olfactory memory'' (enhanced odor differentiation; Sahay et al., 2011b). In fact, a reduction in the generation of olfactory interneurons during adulthood is associated with a decreased ability to discriminate odors (Gheusi et al., 2000). Considering that humans have poor olfactory memory, the role of AHN in olfaction in humans needs to further explored.

#### CREB and Adult Hippocampal Neurogenesis

AHN is a highly regulated process, and several transcription factors have been reported to be involved in this regulation (Hodge and Hevner, 2011). In this regard, the role of CREB in AHN process has been widely investigated. The presence of phosphorylated CREB (pCREB) has been found in many new-born immature neurons in the most important neurogenic niches, including the DG in the SGZ (Nakagawa et al., 2002a,b; Jagasia et al., 2009), SVZ, and OB system (Giachino et al., 2005; Herold et al., 2011). CREB activation in the DG resulting in postnatal hippocampal neurogenesis was first reported in Young et al. (1999). Jagasia et al. (2009) found that CREB expression in the DG persists for 3–21 days after cell generation and overlaps with doublecortin (DCX) expression. DCX is a microtubule-associated protein characteristic of immature neurons and precursor cells. The expression of pCREB subsided with the emergence of mature neuronal markers such as calbindin in mature granule cells (Jagasia et al., 2009) or NeuN in the SVZ/OB neurogenic system, where pCREB presence declines along a rostral-to-caudal gradient (Giachino et al., 2005; Herold et al., 2011).

CREB signaling in adult neurogenesis has been widely studied using in vivo and in vitro approaches. Two groups used rolipram, an inhibitor of phosphodiesterase-4-enhancing CREB signaling, (Nakagawa et al., 2002b; Fujioka et al., 2004) to examine CREB function in vivo; they reported an increase in different parameters of adult neurogenesis in the SGZ, including a higher survival rate of new neurons, more precursor cell proliferation, and enhanced neurite outgrowth and dendritic branching (Merz et al., 2011). CREB/CREM signaling has been described as crucial for survival of adult-born neurons. The positive effects of CREB in AHN were inhibited when CREB expression was repressed. In addition, Giachino et al. (2005) reported a significant decrease in number of newborn cells in the OB of transgenic CREM-null knockout mice. Other studies have shown that rolipram's effects on the morphology of immature and mature neurons in the adult hippocampal DG were eradicated when CREB signaling was repressed by expressing a dominant-negative CREB inhibitor (Fujioka et al., 2004).

The role of CREB signaling in neuronal activation and during survival stages of neurogenesis is better understood than its effects on cell migration and proliferation (Dworkin and Mantamadiotis, 2010). In addition, the pharmacological and genetic approaches used to study the relationship between CREB and AHN do not only affect newborn neurons. Therefore, whether CREB signaling exerts its action by controlling NSCs/new neurons or altering the neurogenic context is an open question warranting further research.

Glucocorticoids are widely considered to negatively influence adult neurogenesis. For example, dexamethasone given at postnatal day 6, a crucial point in the generation of hippocampal neural precursors that survive in the adult brain (Ortega-Martinez, 2015; Ortega-Martínez and Trejo, 2015), suppresses AHN, decreases cell proliferation, and induces behavioral impairments (e.g., learning deficits and increased anxiety; Ortega-Martinez, 2015; Ortega-Martínez and Trejo, 2015). Notably, increased levels of corticosterone, which is known to decrease neural precursor cell proliferation and contribute to neuronal atrophy, were also correlated with lower pCREB levels (Yu et al., 2004).

Various studies have demonstrated that inhibition of CREB signaling with the dominant-negative CREB inhibitor A-CREB (Ahn et al., 1998; Herold et al., 2011) significantly decreases newborn cell survival (Jagasia et al., 2009). New cells showed loss of expression of DCX and Pax6 transcription factor (both of which are related to cell maturity), impairments in rostral migratory stream (RMS) migration, decreased cell proliferation and survival, and increased morphological defects in newborn OB cells (Herold et al., 2011; Merz et al., 2011). The effects of CREB-signaling suppression show that this pathway is crucial for proliferation, differentiation, survival, maturation, and functionality in adult neurogenesis sites (mainly the OB, SVZ, and DG; Merz et al., 2011).

These assumptions are also pertinent to in vivo experiments that have described how CREB signaling affects proliferation of NSC precursors enhancing this cellular mechanism (Kim et al., 2002; Dworkin et al., 2009; Grimm et al., 2009; Merz et al., 2011).

The epigenetic mechanisms of DNA methylation has also been shown to regulate CREB signaling. The CRE sequence contains a CpG island that can be methylated; methylation inhibits binding and restricts CREB localization to functional sites (Zhang et al., 2005). Furthermore, neuronal activity regulates DNA methylation via different enzymes such as DNAdemethylase Gadd5b, suggesting a new link between CREB signaling and neuronal activity, which may be important in adult neurogenesis (Ma et al., 2009).

Another mechanism by which CREB can regulate neurogenesis is by affecting crucial microRNAs (s). miRNAs are ncRNAs (non-coding RNAs) that generally contain 19–23 nucleotides and regulate translation through one of two mechanisms: degradation of target mRNAs or inhibition of the translation machinery (Merz et al., 2011). miRNAs are known to control both precursor cell survival and differentiation in adult neurogenesis. Notably, CREB controls the expression of both MeCP2 (Klein et al., 2007; Chahrour et al., 2008). miRNA-132, which is a member of a cluster of miRNAs enhanced by neuronal activity (Fiore et al., 2009), participates in neuronal morphogenesis, and regulates cognitive capacity (Vo et al., 2005; Hansen et al., 2013). miRNA-134 regulates CREB post-transcriptionally (Gao et al., 2010). In addition, factors known to regulate the proliferation of neural precursor cells (e.g., Wnt; Hirsch et al., 2007; Dworkin and Mantamadiotis, 2010) have been shown to enhance CREB signaling (Chen et al., 2005) in cultured cortical neurons (Vo et al., 2005).

All of the factors described above activate or inhibit CREB signaling, with or without affecting gene expression. Among the molecules related to adult neurogenesis that are also affected by CREB signaling, it is important to highlight the following: BDNF (Kida, 2012; Sable et al., 2014), prolactin (Wang et al., 2013), bcl-2 (Fujii et al., 2014), polysialylated neuronal cell adhesion molecule (PSA-NCAM; Park et al., 2004; NGF; Lim et al., 2014), and cyclin D2 (Kowalczyk et al., 2004; White et al., 2006).

One hypothesis regarding the influence of CREB on adult neurogenesis comes from a recent study that focused on polyunsaturated fatty acid (PUFA)-GPR40-CREB signaling (Yamashima, 2012). The authors showed that primate neurons possess a PUFA-GPR40-CREB-signaling pathway that exerts its action in adult newborn neurons by enhancing the synthesis of BDNF and PSA-NCAM, both of which are implicated in adult neurogenesis (Yamashima, 2012).

A recent review discussed the functional roles of CREB as a positive regulator in the formation and enhancement of memory (Kida and Serita, 2014). Different genetic models used for the study of CREB function were analyzed. Interestingly, some of the findings are in accordance with the new perspective of CREB modulation of memory via its role in adult neurogenesis. In this regard, several transgenic lines of CREB mice such as CREB-VP16, Y134F and DIEDML, which show enhanced STM, also exhibit upregulation of hippocampal BDNF expression, which is related to higher AHN. In addition, microinfusions of BDNF or a BDNF inhibitor into the dorsal hippocampus were associated with improved or impaired STM, respectively. These findings indicate new roles for CREB in learning and memory; CREB plays a regulatory role in STM via the regulation of BDNF expression (Suzuki et al., 2011; Kida and Serita, 2014). Another similar study, Lee et al. (2013) showed that phytoceramide administration enhanced memory via the upregulation of hippocampal pCREB and BDNF expression, which resulted in increased AHN. These results suggest that phytoceramide contributes to memory enhancement and increased BDNF expression, which could lead to increased neurogenesis (Lee et al., 2013).

Finally, links between CREB signaling and neuropsychiatric diseases (e.g., depression) have been reported, and there is evidence that subjects with these diseases also have impairments in AHN (Gass and Riva, 2007). Chronic antidepressant treatment can upregulate hippocampal CREB activity, similar to CREB pathway upregulation in AHN. One study reported that CREBdeficient mice with an antidepressive phenotype also showed neurogenesis enhancement. Compared to wild-type mice, these mice responded more quickly to depression treatment.

# CREB and Memory

One emergent hypothesis regarding the prevention of consolidation of STM into LTM involves the inhibition of proteins or transcription factors. This hypothesis also draws on the relationship between memory consolidation and affective and emotional depression-related circuits (Vogt et al., 2014). The translation of proteins from genes is crucial for memory consolidation (Flexner et al., 1965; Alberini and Kandel, 2015). Different pharmacological approaches have been used to block mRNA and protein synthesis, but this results in an impairment in LTM consolidation without affecting STM (Suzuki et al., 2004; Duvarci et al., 2008; Alberini and Kandel, 2015).

Brunelli et al. (1976) found that serotonin increases the level of cAMP in sensory neurons. It was one of the first reports of an association between cAMP and learning and memory (Brunelli et al., 1976). A decade later, Marc Montminy and L.M. Bilezikjian described, for first time, CREB as a cellular transcription factor that binds the CRE– thereby increasing the transcription of the somatostatin gene (Montminy and Bilezikjian, 1987). The first report of a direct role of CREB transcription factors, downstream of the cAMP pathway, in memory-related synaptic plasticity was in Dash et al. (1990). Dash et al. (1990) demonstrated that, during LTM in Aplysia neurons, PKA activates gene expression via CREB. They could selectively eliminate the long-term process by blocking the binding of CREB1 to its DNA response element. The approach used by Dash et al. (1990) was the microinjection of CRE oligonucleotides into sensory neurons co-cultured with motor neurons. These oligonucleotide bound to the CREB1 protein within the cell and inhibited CREB1. In addition, these oligonucleotide prevented CREB1 from binding to CRE sites in the regulatory regions of cAMP-responsive genes, thus blocking subsequent gene expression. The injection of this oligonucleotide selectively blocked LTM, but did not have an effect on the STM.

A number of more recent studies have demonstrated that CREB is the main element underlying the conversion of STM to LTM (Barco et al., 2003; Kim et al., 2013; Vogt et al., 2014). Several mutant mice phenotypes have been developed to investigate the roles of CREB in learning and memory formation. Walton et al. (1992) study employed a learning and memory task in which mice with a tetracyclinecontrolled transactivator/operator system specifically expressed a dominant-negative inhibitor of CREB activation in CaMKIIαpositive forebrain cells. It was shown that LTM but not STM was impaired. However, these mutant mice did not exhibit a contextual fear-conditioning deficit (Pittenger et al., 2002). In addition, similar results showing that the genetic loss of CREB function impaired LTM but not STM formation has been obtained by other researchers (Bourtchuladze et al., 1994; Kida et al., 2002; Kida and Serita, 2014). Kida (2012) revealed the crucial role of CREB in the consolidation of contextual fear conditioning using tamoxifen-inducible expression of a dominant-negative CREB repressor (Vogt et al., 2014). Furthermore, mutant mice with inhibited CREB activity exhibited deficits in hippocampal late long-term potentiation (L-LTP). Collectively, this evidence indicates that CREB has a central role in these processes and is necessary for both memory consolidation and LTP.

CREB exerts its action as a molecular switch for memory formation (Suzuki et al., 2011). Viral vectors-mediated gene transfer have been widely used to overexpress CREB. Research has shown that the upregulation of CREB activity promotes memory consolidation (Josselyn et al., 2001; Zhou et al., 2009; Alberini and Kandel, 2015). Even from the traditional point of view that CREB enhances LTM, it is thought that the upregulation of CREB-mediated transcription improves STM (Suzuki et al., 2011). It is established that LTM promotion occurs due to enhanced memory consolidation via upregulation of CREB transcriptional activity (Suzuki et al., 2011). In contrast, an increase in basal levels of BDNF, a CREB target gene, is related to improvements in STM. The upregulation of BDNF and CREB activity synergistically improves LTM formation (Suzuki et al., 2011). Therefore, CREB positively controls memory consolidation and is involved in controlling BDNF expression, which also affects memory processes (Suzuki et al., 2011) and is necessary for AHN.

The main CREB target-gene expression related to memory consolidation and LTP are c-fos, activity-regulated cytoskeleton-associated protein (Arc), and BDNF (Miyamoto, 2006; Alberini and Kandel, 2015). Improvements in memory consolidation have also been attributed to overexpression of CREB or Y134F in the amygdala and hippocampus (Josselyn et al., 2001; Restivo et al., 2009; Sekeres et al., 2010). Studies that used viruses to overexpress CREB in the lateral amygdala, which is involved in the fear memory network (Han et al., 2007; Giachero et al., 2015), demonstrated that neurons in this brain area are functionally interrelated (Han et al., 2007). These neurons (i.e., those overexpressing CREB in the lateral amygdala) exhibit greater neuronal excitability (Zhou et al., 2009). These findings indicate that neurons with high levels of CREB activation may contribute to improved memory via involvement in the memory network (Kida and Serita, 2014).

In addition, some neuropsychiatric or neurodegenerative diseases such as depression (Zaninotto et al., 2015), schizophrenia (Burton and Twamley, 2015), and AD (Takeda et al., 2014) are associated with memory loss. In this regard, CREB has been postulated to change the sensitivity of the nucleus accumbens to rewarding and aversive drugs (Bilbao et al., 2014a,b). Because hippocampal CREB expression is upregulated by chronic antidepressant treatment, CREB activity appears to be involved in the pathogenesis and treatment of depression (Gass and Riva, 2007).

Taken together, these studies demonstrate that CREB plays a key role in the formation, consolidation, and enhancement of both STM and LTM. Importantly, in addition to its direct effect in memory processes, CREB may influence AHN directly or through its ability to modulate gene expression. That is, CREB modulates AHN, which affects memory consolidation. CREB mainly contributes to AHN by affecting the expression of genes such as BDNF or PSA-NCAM, and also directly participates in neurogenesis via its signaling in newborn neurons. Moreover, CREB signaling pathways are a target of drug therapies to ameliorate brain disorders associated with cognitive dysfunction (e.g., learning and memory impairments; Kida and Serita, 2014), and some of these drugs are designed to improve memory by stimulating AHN.

# Concluding Remarks Regarding the Implications for CREB's Role in Memory via AHN Regulation

This review has examined how CREB regulates both adult neurogenesis and memory. With regard to the former, enhanced CREB signaling has been widely described to promote newborn neuron survival, precursor cell proliferation, and neurite outgrowth, and dendritic branching (Giachino et al., 2005; Merz et al., 2011). CREB regulation has multiple aspects, including its phosphorylation and recruitment of chromatin remodelers such as CBP and p300, which are essential for CREB gene transcription (Merz et al., 2011) This process is also regulated by multiple cofactors (Merz et al., 2011) facilitating the transcription of different genes involved in AHN mechanisms, such as the expression of BDNF, PSA-NCAM, and NGF2 (**Figure 3**). Furthermore, CREB regulates AHN directly by affecting gene expression and indirectly by influencing epigenetic mechanisms such as inhibitory DNA methylation in its CpG island (Zhang et al., 2005). Neuronal activity controlling DNA methylation patterns (Ma et al., 2009) establish interdependence between these two integrated processes (i.e., neuronal activity and CREB activation). Another mechanism by which CREB can control AHN is by mediating miRNA expression (Vo et al., 2005; Klein et al., 2007; Chahrour et al., 2008; **Figure 3**).

The relationship between CREB and memory consolidation has been widely studied since Brunelli et al. (1976). It is now accepted that CREB is the key molecule responsible for converting STM to LTM and that it plays a central role in memory consolidation (Barco et al., 2003; Kim et al., 2013; Vogt et al., 2014). It has also been established that CREB mainly affects STM by enhancing BDNF gene expression (Suzuki et al., 2011). Finally, hippocampal CREB overexpression improves memory formation (Restivo et al., 2009; Sekeres et al., 2010).

Taken together, the existing literature suggests that CREB enhances memory through several different mechanisms. Directly, it can increase expression of genes that modulate memory. The new perspective discussed here is that CREB modulates AHN, which also affect memory processes. In this regard, CREB enhances the expression of target genes such as BDNF and PSA-NCAM, which augment AHN. In addition, CREB upregulation itself promotes AHN, leading to greater neuronal survival and postnatal hippocampal neurogenesis (Cameron and Glover, 2015). CREB was also recently shown to be crucial for AHN. AHN has a central role in several cognitive processes including spatial, recognition, and fear conditioning memory (Cameron and Glover, 2015; **Figure 3**). Based on the evidence, it is reasonable to formulate the novel hypothesis referred to here as ''A new perspective of memory regulation based on CREB-mediated hippocampal neurogenesis'' in which CREB controls memory processes by direct regulation and effects on adult neurogenesis, which affects cognitive and memory processes.

In addition, it is important to not underscore that cognitive process attributable to CREB transcription factors are complex and difficult to study. In this regard, evidence for this hypothesis is crucial to design follow-up studies. Even though there is considerable accumulated knowledge in this domain, it is necessary to better understand how CREB works in adult neurogenesis. Many questions remain unanswered. For example, can CREB also modulate the neurogenic niche or directly affect new neurons? It would be interesting to clarify whether CREB can modify the SGZ microenvironment to facilitate AHN. Another important question is whether CREB can modulate or change the phenotype of a specific neural lineage (e.g., stem or progenitor cells instead of mature neurons). Appropriate gene expression is crucial in determining phenotype, and the transcription of different genes affects diverse mechanisms including cell quiescence, proliferation, and death. Finally, can CREB modulate adult neurogenesis through the control of other adult neurogenesis regulators such as neurotransmitters, steroid hormones, or cytokines (Ortega-Martinez, 2015)?

Importantly, due to its effect on AHN, CREB may be a strategic target in the development of therapeutic drugs for neurodegenerative and psychiatric diseases associated with cognitive impairment.

# Author Contributions

SO-M is the sole author of this paper and is responsible for its content.

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### Acknowledgments

This work was supported by grant to Dr. Eleanor Coffey from the FP7 Marie Curie ITN r'BIRTH. The author acknowledges Marie Curie fellowship and Åbo Akademi University for supporting SO-M. The author is grateful Dr. Eleanor Coffey for the opportunity to work in her lab and for her help and support. SO-M received a postdoctoral fellowship from Marie Curie ITN r'BIRTH.

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**Conflict of Interest Statement**: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.