Vorinostat, a histone deacetylase inhibitor, facilitates fear extinction and enhances expression of the hippocampal NR2B-containing NMDA receptor gene

Abstract

Histone acetylation, which alters the compact chromatin structure and changes the accessibility of DNA to regulatory proteins, is emerging as a fundamental mechanism for regulating gene expression. Histone deacetylase (HDAC) inhibitors increase histone acetylation and enhance fear extinction. In this study, we examined whether vorinostat, an HDAC inhibitor, facilitates fear extinction, using a contextual fear conditioning (FC) paradigm, in Sprague-Dawley rats. We found that vorinostat facilitated fear extinction. Next, the levels of global acetylated histone H3 and H4 were measured by Western blotting. We also assessed the effect of vorinostat on the hippocampal levels of NMDA receptor mRNA by real-time quantitative PCR (RT-PCR) and protein by Western blotting. 2 h after vorinostat administration, the levels acetylated histones and NR2B mRNA, but not NR1 or NR2A mRNA, were elevated in the hippocampus. The NR2B protein level was elevated 4 h after vorinostat administration. Last, we investigated the levels of acetylated histones and phospho-CREB (p-CREB) binding at the promoter of the NR2B gene using the chromatin immunoprecipitation (ChIP) assay followed by RT-PCR. The ChIP assay revealed increases in the levels of acetylated histones and they were accompanied by enhanced binding of p-CREB to its binding site at the promoter of the NR2B gene 2 h after vorinostat administration. These findings suggest that vorinostat increases the expression of NR2B in the hippocampus by enhancing histone acetylation, and this process may be implicated in fear extinction.

Introduction

At present, selective serotonin reuptake inhibitors (SSRIs) or benzodiazepines are the primary pharmacologic treatments for human anxiety disorders such as posttraumatic stress disorder (PTSD) and phobias; however, their therapeutic efficacy has not been well-established. It has been proposed that impairment of fear extinction plays a pivotal role in the pathophysiology of PTSD (Kaplan and Moore, 2011; Myers and Davis, 2007). As a consequence, drugs that facilitate fear extinction may be useful as novel treatments for PTSD. The N-methyl d-aspartate (NMDA) receptor may play an important role in learning and memory and in experience-dependent forms of plasticity such as long-term potentiation (LTP) (Nicoll and Malenka, 1999). In addition, several studies have shown cognitive-enhancing effects of NMDA receptor agonists, such as d-cycloserine (DCS), on fear extinction (Davis et al., 2006; Ledgerwood et al., 2005; Walker and Davis, 2002). Furthermore, Ressler et al. (2004) and Hofmann et al. (2006) reported that DCS facilitates fear extinction in patients with anxiety disorders.

Fear memory extinction requires memory consolidation mediated via gene transcription (Myers and Davis, 2002). Transcription is regulated by the concerted action of transcription factors and cofactors that modify and remodel the structure of chromatin (Holliday, 2006). Thus, a clear understanding of the epigenetic mechanisms of fear extinction and associated alterations in gene expression may provide opportunities for the development of novel therapies. Histone acetylation, which alters the compact chromatin structure and changes the accessibility of DNA to regulatory proteins, is emerging as a fundamental mechanism for regulating gene expression (Goldberg et al., 2007; Kurdistani and Grunstein, 2003). Histone acetylation is regulated by the opposing activities of histone acetyltransferases (HAT) and histone deacetylases (HDAC) (Kramer et al., 2001).

Several recent studies have demonstrated that histone acetylation plays a role in learning behavior and synaptic plasticity (Fischer et al., 2010; Haettig et al., 2011; Levenson and Sweatt, 2005). Levenson et al. (2004) demonstrated that increased acetylation of histone H3 was correlated with long-term memory formation, and HDAC inhibitors, at least in part, contributed to the induction of LTP and long-term memory. Similarly, Korzus et al. (2004) reported that a decrease in the activity of HAT disturbed the formation of long-term memory, and that the administration of HDAC inhibitors ameliorated this disturbance. Lattal et al. (2007) showed that administration of HDAC inhibitors, such as sodium butyrate (NaB) systemically or trichostatin A (TSA) intra-hippocampally, prior to a contextual extinction session produced greater effects than vehicle treatment in context-evoked fear extinction. Bredy et al. (2007) reported that valproic acid (VPA) enhanced fear extinction through histone H4 acetylation around the promoter region of the brain-derived neurotrophic factor (BDNF) gene and also increased BDNF mRNA expression in the prefrontal cortex.

Vorinostat, an HDAC inhibitor, has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of refractory cutaneous T-cell lymphoma under the trade name Zolinza^®. Although vorinostat was primarily studied in the treatment of cancer, it has recently been applied in the preclinical development of a novel treatment regimen for memory disturbance. For example, chronic or acute injections of vorinostat into specific brain regions have been shown to increase synaptic plasticity and facilitate memory formation in mice (Guan et al., 2009; Peleg et al., 2010). Administration of vorinostat was also found to increase synaptic plasticity and learning behavior in an animal model of Rubinstein–Taybi syndrome (RTS), an inheritable disorder caused by mutations in the gene encoding the cyclic AMP-response element-binding protein (CREB) binding protein (CBP) and characterized by mental retardation and skeletal abnormalities. Mice lacking CBP (CBP+/− mice) recapitulate the major phenotypes of RTS. Vorinostat treatment of CBP+/− mice was correlated with elevated histone acetylation and improved memory function (Alarcon et al., 2004). Vorinostat was reported to be brain-permeable (Palmieri et al., 2009), and daily systemic administration of vorinostat for 19 days was found to rescue cognitive deficits in a mouse model of Alzheimer's disease (APPswe/PS1dE9 mice) (Kilgore et al., 2009). However, little is known about the effect of vorinostat on fear extinction.

In the present study, we first examined whether vorinostat enhances fear extinction, using a contextual fear conditioning (FC) paradigm. Further, we investigated the effects of vorinostat on some behavioral aspects. We also assessed the effect of vorinostat on the hippocampal levels of NMDA receptor mRNA and protein. To elucidate whether the up-regulation of the NR2B gene in the hippocampus is due to the enhancement of histone acetylation by vorinostat, we determined the levels of global acetylated histones by Western blotting, and the degree of histone acetylation at the promoter region of the NR2B gene in the hippocampus by chromatin immunoprecipitation (ChIP) assay followed by real-time quantitative polymerase chain reaction (RT-PCR). In addition, we examined whether vorinostat increases the binding of phospho-CREB (p-CREB) to the cyclic AMP-response element (CRE) site at the promoter of the NR2B gene using ChIP assay followed by RT-PCR.