Marcelo A. Wood

Histone deacetylase inhibitors enhance memory and synaptic plasticity via CREB: CBP-dependent transcriptional activation

Histone deacetylase (HDAC) inhibitors increase histone acetylation and enhance both memory and synaptic plasticity. The current model for the action of HDAC inhibitors assumes that they alter gene expression globally and thus affect memory processes in a nonspecific manner. Here, we show that the enhancement of hippocampus-dependent memory and hippocampal synaptic plasticity by HDAC inhibitors is mediated by the transcription factor cAMP response element-binding protein (CREB) and the recruitment of the transcriptional coactivator and histone acetyltransferase CREB-binding protein (CBP) via the CREB-binding domain of CBP. Furthermore, we show that the HDAC inhibitor trichostatin A does not globally alter gene expression but instead increases the expression of specific genes during memory consolidation. Our results suggest that HDAC inhibitors enhance memory processes by the activation of key genes regulated by the CREB:CBP transcriptional complex.

HDAC3 Is a Critical Negative Regulator of Long-Term Memory Formation

Gene expression is dynamically regulated by chromatin modifications on histone tails, such as acetylation. In general, histone acetylation promotes transcription, whereas histone deacetylation negatively regulates transcription. The interplay between histone acetyltranserases and histone deacetylases (HDACs) is pivotal for the regulation of gene expression required for long-term memory processes. Currently, very little is known about the role of individual HDACs in learning and memory. We examined the role of HDAC3 in long-term memory using a combined genetic and pharmacologic approach. We used HDAC3–FLOX genetically modified mice in combination with adeno-associated virus-expressing Cre recombinase to generate focal homozygous deletions of Hdac3 in area CA1 of the dorsal hippocampus. To complement this approach, we also used a selective inhibitor of HDAC3, RGFP136 [N-(6-(2-amino-4-fluorophenylamino)-6-oxohexyl)-4-methylbenzamide]. Immunohistochemistry showed that focal deletion or intrahippocampal delivery of RGFP136 resulted in increased histone acetylation. Both the focal deletion of HDAC3 as well as HDAC3 inhibition via RGFP136 significantly enhanced long-term memory in a persistent manner. Next we examined expression of genes implicated in long-term memory from dorsal hippocampal punches using quantitative reverse transcription-PCR. Expression of nuclear receptor subfamily 4 group A, member 2 (Nr4a2) and c-fos was significantly increased in the hippocampus of HDAC3–FLOX mice compared with wild-type controls. Memory enhancements observed in HDAC3–FLOX mice were abolished by intrahippocampal delivery of Nr4a2 small interfering RNA, suggesting a mechanism by which HDAC3 negatively regulates memory formation. Together, these findings demonstrate a critical role for HDAC3 in the molecular mechanisms underlying long-term memory formation.

Modulation of long-term memory for object recognition via HDAC inhibition

Histone acetylation is a chromatin modification critically involved in gene regulation during many neural processes. The enzymes that regulate levels of histone acetylation are histone acetyltransferases (HATs), which activate gene expression and histone deacetylases (HDACs), that repress gene expression. Acetylation together with other histone and DNA modifications regulate transcription profiles for specific cellular functions. Our previous research has demonstrated a pivotal role for cyclicAMP response element binding protein (CREB)-binding protein (CBP), a histone acetyltransferase, in long-term memory for novel object recognition (NOR). In fact, every genetically modifiedCbp mutant mouse characterized thus far exhibits impaired long-term memory for NOR. These results suggest that long-term memory for NOR is especially sensitive to alterations in CBP activity. Thus, in the current study, we examined the role of HDACs in memory for NOR. We found that inducing a histone hyperacetylated state via HDAC inhibition transforms a learning event that would not normally result in long-term memory into an event that is now remembered long-term. We have also found that HDAC inhibition generates a type of long-term memory that persists beyond a point at which normal memory for NOR fails. This result is particularly interesting because one alluring aspect of examining the role of chromatin modifications in modulating transcription required for long-term memory processes is that these modifications may provide potentially stable epigenetic markers in the service of activating and/or maintaining transcriptional processes.

Systemic or intrahippocampal delivery of histone deacetylase inhibitors facilitates fear extinction.

Several recent studies have shown that chromatin, the DNA-protein complex that packages genomic DNA, has an important function in learning and memory. Dynamic chromatin modification via histone deacetylase (HDAC) inhibitors and histone acetyltransferases may enhance hippocampal synaptic plasticity and hippocampus-dependent memory. Little is known about the effects of HDAC inhibitors on extinction, a learning process through which the ability of a previously conditioned stimulus, such as a conditioning context, to evoke a conditioned response is diminished. The authors demonstrate that administration of the HDAC inhibitors sodium butyrate (NaB) systemically or trichostatin A (TSA) intrahippocampally prior to a brief (3-min) contextual extinction session causes context-evoked fear to decrease to levels observed with a long (24-min) extinction session. These results suggest that HDAC inhibitors may enhance learning during extinction and are consistent with other studies demonstrating a role for the hippocampus in contextual extinction. Molecular and behavioral mechanisms through which this enhanced extinction effect may occur are discussed.

Beyond transcription factors: The role of chromatin modifying enzymes in regulating transcription required for memory

One of the alluring aspects of examining chromatin modifications in the role of modulating transcription required for long-term memory processes is that these modifications may provide transient and potentially stable epigenetic marks in the service of activating and/or maintaining transcriptional processes. These, in turn, may ultimately participate in the molecular mechanisms required for neuronal changes subserving long-lasting changes in behavior. As an epigenetic mechanism of transcriptional control, chromatin modification has been shown to participate in maintaining cellular memory (e.g., cell fate) and may underlie the strengthening and maintenance of synaptic connections required for long-term changes in behavior. Epigenetics has become central to several fields of neurobiology, where researchers have found that regulation of chromatin modification has a significant role in epilepsy, drug addiction, depression, neurodegenerative diseases, and memory. In this review, we will discuss the role of chromatin modifying enzymes in memory processes, as well as how recent studies in yeast genetics and cancer biology may impact the way we think about how chromatin modification and chromatin remodeling regulate neuronal function.

HDAC3-selective inhibitor enhances extinction of cocaine-seeking behavior in a persistent manner.

Nonspecific histone deacetylase (HDAC) inhibition has been shown to facilitate the extinction of drug-seeking behavior in a manner resistant to reinstatement. A key open question is which specific HDAC is involved in the extinction of drug-seeking behavior. Using the selective HDAC3 inhibitor RGFP966, we investigated the role of HDAC3 in extinction and found that systemic treatment with RGFP966 facilitates extinction in mice in a manner resistant to reinstatement. We also investigated whether the facilitated extinction is related to the enhancement of extinction consolidation during extinction learning or to negative effects on performance or reconsolidation. These are key distinctions with regard to any compound being used to modulate extinction, because a more rapid decrease in a defined behavior is interpreted as facilitated extinction. Using an innovative combination of behavioral paradigms, we found that a single treatment of RGFP966 enhances extinction of a previously established cocaine-conditioned place preference, while simultaneously enhancing long-term object-location memory within subjects. During extinction consolidation, HDAC3 inhibition promotes a distinct pattern of histone acetylation linked to gene expression within the infralimbic cortex, hippocampus, and nucleus accumbens. Thus, the facilitated extinction of drug-seeking cannot be explained by adverse effects on performance. These results demonstrate that HDAC3 inhibition enhances the memory processes involved in extinction of drug-seeking behavior.

Epigenetics in the Nervous System

It is becoming increasingly clear that epigenetic modifications are critical factors in the regulation of gene expression. With regard to the nervous system, epigenetic alterations play a role in a diverse set of processes and have been implicated in a variety of disorders. Gaining a more complete understanding of the essential components and underlying mechanisms involved in epigenetic regulation could lead to novel treatments for a number of neurological and psychiatric conditions.

Hippocampal Focal Knockout of CBP Affects Specific Histone Modifications, Long-Term Potentiation, and Long-Term Memory

To identify the role of the histone acetyltransferase (HAT) CREB-binding protein (CBP) in neurons of the CA1 region of the hippocampus during memory formation, we examine the effects of a focal homozygous knockout of CBP on histone modifications, gene expression, synaptic plasticity, and long-term memory. We show that CBP is critical for the in vivo acetylation of lysines on histones H2B, H3, and H4. CBPs homolog p300 was unable to compensate for the loss of CBP. Neurons lacking CBP maintained phosphorylation of the transcription factor CREB, yet failed to activate CREB:CBP-mediated gene expression. Loss of CBP in dorsal CA1 of the hippocampus resulted in selective impairments to long-term potentiation and long-term memory for contextual fear and object recognition. Together, these results suggest a necessary role for specific chromatin modifications, selectively mediated by CBP in the consolidation of memories.

HDAC inhibition modulates hippocampus-dependent long-term memory for object location in a CBP-dependent manner

Transcription of genes required for long-term memory not only involves transcription factors, but also enzymatic protein complexes that modify chromatin structure. Chromatin-modifying enzymes, such as the histone acetyltransferase (HAT) CREB (cyclic-AMP response element binding) binding protein (CBP), are pivotal for the transcriptional regulation required for long-term memory. Several studies have shown that CBP and histone acetylation are necessary for hippocampus-dependent long-term memory and hippocampal long-term potentiation (LTP). Importantly, every genetically modified Cbp mutant mouse exhibits long-term memory impairments in object recognition. However, the role of the hippocampus in object recognition is controversial. To better understand how chromatin-modifying enzymes modulate long-term memory for object recognition, we first examined the role of the hippocampus in retrieval of long-term memory for object recognition or object location. Muscimol inactivation of the dorsal hippocampus prior to retrieval had no effect on long-term memory for object recognition, but completely blocked long-term memory for object location. This was consistent with experiments showing that muscimol inactivation of the hippocampus had no effect on long-term memory for the object itself, supporting the idea that the hippocampus encodes spatial information about an object (such as location or context), whereas cortical areas (such as the perirhinal or insular cortex) encode information about the object itself. Using location-dependent object recognition tasks that engage the hippocampus, we demonstrate that CBP is essential for the modulation of long-term memory via HDAC inhibition. Together, these results indicate that HDAC inhibition modulates memory in the hippocampus via CBP and that different brain regions utilize different chromatin-modifying enzymes to regulate learning and memory.

Modulation of Chromatin Modification Facilitates Extinction of Cocaine-Induced Conditioned Place Preference

BACKGROUND Recent evidence suggests that epigenetic mechanisms have an important role in the development of addictive behavior. However, little is known about the role of epigenetic mechanisms in the extinction of drug-induced behavioral changes. In this study, we examined the ability of histone deacetylase (HDAC) inhibitors to facilitate extinction and attenuate reinstatement of cocaine-induced conditioned place preference (CPP). METHODS C57BL/6 mice were subject to cocaine-induced CPP using 20 mg/kg dose. To facilitate extinction, mice were administered an HDAC inhibitor following nonreinforced exposure to the conditioned context. To measure persistence, mice were subject to a reinstatement test using 10 mg/kg dose of cocaine. RESULTS We demonstrate that HDAC inhibition during extinction consolidation can facilitate extinction of cocaine-induced CPP. Animals treated with an HDAC inhibitor extinguished cocaine-induced CPP both more quickly and to a greater extent than did vehicle-treated animals. We also show that the extinction of cocaine seeking via HDAC inhibition modulates extinction learning such that reinstatement behavior is significantly attenuated. Acetylation of histone H3 in the nucleus accumbens following extinction was increased by HDAC inhibition. CONCLUSIONS This study provides the first evidence that modulation of chromatin modification can facilitate extinction and prevent reinstatement of drug-induced behavioral changes. These findings provide a potential novel approach to the development of treatments that facilitate extinction of drug-seeking behavior.