The results from this study demonstrate that HDAC3 is a critical negative regulator of long-term memory formation. Focal homozygous gene deletion of HDAC3 resulted in not only loss of HDAC3 but also a significant decrease in HDAC4 expression. Neurons lacking HDAC3 had increased histone acetylation of histone H4 lysine 8 (H4K8Ac), which correlated with increased Nr4a2 and c-fos expression in the area of the focal HDAC3 deletion in the dorsal hippocampus. Focal homozygous deletion of HDAC3 in the dorsal hippocampus lead to facilitated long-term memory formation after a subthreshold training period. This subthreshold training period failed to yield long-term memory in control animals. The genetic approach to examine the role of HDAC3 in long-term memory formation was complemented with a pharmacological approach using an HDAC inhibitor shown to be more selective for HDAC3 than other class I HDACs. This compound, called RGFP136, when delivered to the dorsal hippocampus, resulted in decreased HDAC4 expression, increased H4K8Ac, and also significantly facilitated long-term memory formation via a CBP-dependent manner in the hippocampus. Our final experiment demonstrated that HDAC3 may modulate long-term memory formation via the expression of the immediate early gene and transcription factor Nr4a2. Together, these genetic and neuropharmacological approaches identify HDAC3 as a critical negative regulator of memory.
HDAC3 is expressed throughout the brain, with particularly strong gene expression in the hippocampus (Broide et al., 2007
). However, no study to date has examined the role of HDAC3 in the brain. Previous in vitro
studies have shown that HDAC3 and HDAC4 interact with each other in large complexes (Grozinger and Schreiber, 2000
; Fischle et al., 2002
). Interactions between HDAC3 and HDAC4 create a functional complex involved in transcriptional regulation. HDAC4 is considered to be in the “inactive state” until bound to HDAC3, an interaction necessary for its enzymatic activity (Fischle et al., 2002
). A study by Lahm et al. (2007)
supported previous findings that class IIa HDACs (HDAC4, HDAC5, HDAC7, and HDAC9) are inactive on acetylated substrates, thus distinguishing them from class I HDACs (HDAC1, HDAC2, HDAC3, and HDAC8). This has called into question the catalytic activity of class IIa HDACs. An equally reasonable idea is that the natural substrate of these enzymes has not been identified. In any case, the interaction between HDAC4 and HDAC3 is facilitated by corepressor proteins NCoR and SMRT, which form a large complex with HDACs and other proteins (Fischle et al., 2002
). HDAC4 and HDAC3 bind independently to different domains of SMRT and NCoR, but the proximity allows for interactions of HDAC3 and HDAC4 proteins.
To complement our genetic and pharmacological approach to study HDAC3, we also used genetically modified NCoR mutant mice. These mice, referred to as DADm mice, carry a single amino acid substitution (Y478A) in the DAD of NCoR, which results in a mutant NCoR protein that is unable to associate with or activate HDAC3 (Guenther et al., 2001
; Ishizuka and Lazar, 2005
; Alenghat et al., 2008
). When given a subthreshold training period, DADm homozygous knock-in mice exhibited significant long-term memory compared with wild-type littermates, which failed to show any long-term memory. These data support the idea that a functional complex between NCoR and HDAC3 is required to repress long-term memory formation.
Lahm et al. (2007)
showed that a critical residue for HDAC3 activity is a tyrosine at amino acid 298, which if mutated to a histidine (Y298H) completely abrogates enzymatic function. Interestingly, HDAC4 and other class IIa enzymes normally have a histidine at this position, which provides a potential reason why HDAC4 has such poor enzymatic activity on traditional substrates. Commonly used HDAC inhibitors, such as valproic acid, sodium butyrate, phenylbutyrate, and suberoylanilide hydroxamic acid (SAHA), have been shown to greatly inhibit class I HDACs (HDAC1, HDAC2, HDAC3, HDAC8) with little effect on the class IIa HDAC family members (HDAC4, HDAC5, HDAC7, HDAC9) (Kilgore et al., 2010
). This suggests that inhibition of class I HDACs are critical for the reported effects of HDAC inhibition, such as the enhancement of cognition. Indeed, HDAC2, which has been shown to negatively regulate memory formation, has been implicated as a specific target of commonly used HDAC inhibitors (Guan et al., 2009
Recently, a new class of HDAC inhibitor called pimelic diphenylamide compounds has been identified (Chou et al., 2008
; Xu et al., 2009
; Rai et al., 2010
). These inhibitors are slow-on/slow-off, competitive tight-binding inhibitors that specifically target class I HDACs, with the greatest inhibitory effect on HDAC3 (Chou et al., 2008
; Xu et al., 2009
). RGFP136, used in these studies, has an IC50
of 5.2 μm
for HDAC1, 3.0 μm
for HDAC2, and 0.4 μm
for HDAC3 using purified recombinant HDACs. After systemic subcutaneous injection, the maximum drug concentration (Cmax
) in the brain is ~1.7 μm
for a 30 mg/kg dose. This suggests that, after systemic administration, as in the data shown in , RGFP136 is at a sufficient concentration in the brain to inhibit HDAC3 but perhaps not HDAC1 or HDAC2. Furthermore, the immunofluorescence data indicate that RGFP136 disrupts HDAC4 expression, with no effect on HDAC2 expression. Thus, although RGFP136 affects class I HDACs, the effects observed in this study are most likely via HDAC3. Behaviorally, when delivered site specifically to the dorsal hippocampus, RGFP136 transformed a learning event that does not result in long-term memory into an event that now does lead to long-term memory. Furthermore, this facilitation of long-term memory via RGFP136 resulted in persistent long-term memory observed 7 d later when normal long-term memory retrieval for object location fails. Subcutaneous injection of RGFP136 also facilitated long-term memory for object location () as well as long-term memory for a familiar object ().
Importantly, we found that, in the hippocampus, RGFP136 requires CBP to facilitate long-term memory formation. CBPKIX/KIX
mice, which contain a mutation in the phospho-CREB (KIX) binding domain of CBP (Kaspar et al., 2002
), failed to exhibit significant long-term memory for object location when RGFP136 was delivered to the dorsal hippocampus (). These results suggest that RGFP136 is functioning via a CBP-dependent mechanism to regulate transcription required for hippocampus-dependent long-term memory.
Despite the consistent enhancements of long-term memory by deletion of HDAC3, short-term memory was unaffected (, ). A major difference between these forms of memory is that, in general, transcription is essential for the formation of long-term memory but not short-term memory. We and others have found that HDAC inhibition by sodium butyrate, trichostatin A (TSA), or SAHA have no effect on short-term memory (Korzus et al., 2004
; Levenson et al., 2004
; Yeh et al., 2004
; Vecsey et al., 2007
; Stefanko et al., 2009
). Additionally, homozygous knock-in NCoR mice in this study exhibit enhanced long-term memory but not enhanced short-term memory. In contrast, Guan et al. (2009)
found enhanced short-term memory in HDAC2 knock-out mice in which the knock-out is generated by crossing HDAC2–FLOX mice with nestin–Cre transgenic mice. Thus, the differences observed on short-term memory may be attributable to either functional differences between HDAC3 and HDAC2 or developmental/compensation effects in HDAC2 knock-out mice. In any case, it is still remarkable that traditional knock-out (HDAC2) or knock-in (NCoR) mice exhibit similar enhanced long-term memory phenotypes as acute disruption of HDAC activity by pharmacological manipulation.
A major finding in this study is the relationship of hippocampal HDAC3 deletion with increased Nr4a2
is a CREB-dependent gene that has been implicated in long-term memory (Peña de Ortiz et al., 2000
; von Hertzen and Giese, 2005
; Colón-Cesario et al., 2006
; Vecsey et al., 2007
). We have demonstrated previously that Nr4a2
expression is enhanced by the HDAC inhibitor TSA during memory consolidation (Vecsey et al., 2007
). In this study, we also observed enhanced Nr4a2
expression in HDAC3flox/flox
mice after learning (). It has been suggested that HDACs may terminate the CREB-dependent transcription for this gene (Fass et al., 2003
), and thus the removal of HDAC3 allows transcription to be maintained for a longer period. Activation of Nr4a2
is critical for the expression of long-term memory, as demonstrated by our behavioral study using siRNA (). HDAC3flox/flox
mice with a homozygous deletion of HDAC3 in the dorsal hippocampus failed to exhibit enhanced long-term memory when Nr4a2
siRNA was infused into the area of HDAC3 deletion before training. These findings are in agreement with another study that found hippocampal infusions of Nr4a2
antisense, which did not affect c-fos
expression, caused impaired long-term memory for a spatial discrimination task (Colón-Cesario et al., 2006
). These data suggest a mechanism by which the loss of HDAC3 enhances long-term memory by allowing increased and/or prolonged CREB/CBP-dependent transcription of Nr4a2
In summary, the experiments presented here demonstrate that HDAC3 is a critical negative regulator of long-term memory formation. RGFP136, a pimelic diphenylamide compound, represents a promising pharmacotherapeutic approach for cognitive impairments. RGFP136 and genetic manipulation of HDAC3 (via HDAC3flox/flox and DADm mice) had similar effects at the molecular and behavioral levels. It is likely that HDAC3 performs its role in memory processes via its interactions with NCoR as well as HDAC4. However, future studies are necessary to determine the exact nature of these interactions and their effects on acetylation, gene expression, and learning and memory.