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Effects of Nicotine and Histone Deacetylase Inhibitors on the Brain
C H A P T E R
45 Effects of Nicotine and Histone Deacetylase Inhibitors on the Brain Maria Paula Faillace, Ramon O. Bernabeu Departamento de Fisiologı´a e Instituto de Fisiologı´a y Biofı´sica (IFIBIO-Houssay, UBA-CONICET), Universidad de Buenos Aires (UBA), Paraguay 2155, Buenos Aires, Argentina
Abbreviations CPA CpG CPP CREB DNMT dStr H3 H4 HAT HDAC HDACi NaB Nacc nAChR NOR OR pCREB PFC PhB PrL TSA VTA
conditioning place aversion cytosine-guanine island conditioning place preference cyclic-AMP-responsive element-binding protein DNA methyltransferase dorsal striatum histone 3 histone 4 histone acetyltransferase histone deacetylase HDAC inhibitor sodium butyrate nucleus accumbens nicotinic acetylcholine receptor novel object recognition object recognition phosphorylated CREB prefrontal cortex phenylbutyrate prelimbic frontal area trichostatin A ventral tegmental area
45.1 INTRODUCTION Genetic information is stored in extremely long linear sequences of nucleotides, so the genetic material in eukaryotes is arranged into complex structures, which efficiently pack DNA within the cell nucleus (Horn & Peterson, 2002). DNA chains together with nuclear proteins form the chromatin. The histones (H) are the chief structural and constitutive regulatory proteins in chromatin. One hundred and forty-six base pairs of DNA are wrapped twice around an octamer of histones. Each octamer consists of two subunits of each type of histone: H2A, H2B, H3, and H4. This basic structural unit of chromatin is termed nucleosome.
Neuroscience of Nicotine https://doi.org/10.1016/B978-0-12-813035-3.00045-9
Epigenesis involves effects determined by a huge array of chromatin changes, which are principally induced by DNA methylation and histone postranslational modifications ( Jiang et al., 2008). Other processes can also control gene transcription by chromatin structural remodeling, and all epigenetic changes are heritable and reversible. However, no changes in DNA or protein sequence occur during epigenetic regulation. DNA methylation is the core of long-term epigenetic changes, and for several years, DNA methylation was conceived only as cytosine methylation. However, a few years ago, it was found that guanine and adenine can be methylated (O’Brown & Greer, 2016). Furthermore, histone enzymatic modifications are the most common and studied epigenetic changes that can induce long- and short-term changes in the chromatin structure. Histones have a tail that extends out of the octamer core of the nucleosome. The amino-terminal tails undergo postranslational modifications including acetylation, methylation, phosphorylation, ADP-ribosylation, farnesylation, proline isomerization, and ubiquitination (Tollefsbol, 2011). These chemical groups modify histone-DNA and histone-protein interactions remodeling chromatin with consequences for transcription and DNA replication and repair ( Jenuwein & Allis, 2001). Hence, gene expression can be driven by the acetylation and methylation of histones, which modifies DNA-protein associations and chromatin structure (Fig. 45.1). Chromatin relaxed or condensed forms favor or reduce transcription, respectively. The acetylation and demethylation of residues in histones induce DNA relaxation and promote transcription factor and remodeling protein interaction (Kouzarides, 2007; Marmorstein & Zhou, 2014). In this chapter, we analyzed epigenetic mechanisms that involve histone acetylation. Moreover, we described published results about histone deacetylase inhibitor
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Copyright © 2019 Elsevier Inc. All rights reserved.
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FIG. 45.1 Histone acetylation and deacetylation are epigenetic mechanisms, which provoke opposite effects on chromatin structure and gene expression. Cartoon representing the epigenetic mechanism of histone acetylation of lysine residues located at histone 3 and 4 (H3 and H4) tails. Acetylation and deacetylation of histones mediate conversion between relaxed and condensed chromatin forms, respectively. (A) Histone tails are fully acetylated that induces an open state of the chromatin, which is more accessible to transcription factor interaction promoting transcription. The acetylated state is promoted by histone acetyltransferase activity. (B) Histone tail deacetylation condenses chromatin, which reduces transcription. The removal of acetyl groups from lysine residues is achieved by histone deacetylase activity. (C) Intermediate acetylated states promote a heterogeneous chromatin structure that differentially regulates transcription. Histone deacetylase inhibitors (HDACi) induce higher degrees of acetylation generally favoring gene expression. Epigenetic regulators such as nicotine can induce acetylation of specific portions of the chromatin differentially regulating gene expression. “Unpublished and original art work by R. O. Bernabeu.”
(HDACi) effects on animal behavior and protein expression related to nicotine rewarding properties.
45.2 HISTONE ACETYLATION AND DEACETYLATION BY ACETYLTRANSFERASES AND DEACETYLASES Many factors from the environment can activate the enzymes that regulate histone modifications. Histone acetylation plays a prominent role affecting chromatin structure. Acetylation of histones is a dynamic and reversible process controlled by a family of enzymes called histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs transfer acetyl groups to lysine residues, and HDACs remove acetyl groups from these amino acids. The first HAT identified called Hatl was found in yeast in 1995, and 1 year later, the first HDAC
was purified and termed HDAC-l (Yang & Seto, 2007). HAT and HDAC activation provides a mechanism to understand how histone acetylation and transcription are regulated. For instance, studies dealing with type A HATs (GCN5, p300/CBP, TAFII250, p55, and PCAF) provided information about acetylation of nucleosome histones, whereas studies dealing with type B HATs informed about acetylation mechanisms of newly synthesized histones in the cytoplasm ( Jeanteur, 2005).
45.2.1 Histone Deacetylase Inhibitors HDAC inhibitors maintain histone acetylation and can tip the balance to an “open” configuration state of chromatin leading to a transcriptional permissive state. Different drugs characterized as HDAC inhibitors have been used for the treatment of neuropsychiatric and drug addiction diseases. For instance, valproate is an HDAC inhibitor that has been extensively used for treating
45.2 HISTONE ACETYLATION AND DEACETYLATION BY ACETYLTRANSFERASES AND DEACETYLASES
neuropsychiatric disorders. Valproate induces DNA demethylation in the adult mouse brain (Dong, Chen, Gavin, Grayson, & Guidotti, 2010). Likewise, trichostatin A (TSA) increases histone acetylation and decreases DNA methylation. However, treatments with an inhibitor of DNA methyltransferases did not affect histone acetylation suggesting that acetylation can modulate methylation but not the opposite (Vaissière, Sawan, & Herceg, 2008). SIRT1 (class III HDAC) deacetylates DNA methyltransferase 1 (DNMT1), so it is likely that HDAC activity inhibitors regulate DNA methylation levels by modifying a key methyltransferase activity (Peng et al., 2011). Furthermore, the administration of HDAC inhibitors at the appropriate time point alleviates the negative dysphoric effects observed in alcohol withdrawal (Sakharkar et al., 2014). At present, more than 40 drugs that work as HDAC inhibitors have been described. Some of them were approved by the Food and Drug Administration (FDA) and are principally used to treat different cancer types, whereas other compounds are waiting for approval for clinical use. In this chapter, we focused in two HDAC inhibitors: sodium butyrate (NaB) and phenylbutyrate (PhB), which have demonstrated significant biological and behavioral effects on animal models for the study of nicotine dependence.
45.2.2 Histone Deacetylase and Nicotine Effects on the Nervous System 45.2.2.1 Nicotine Mimics the Action of HDAC Inhibitors Consumption of abused drugs, including nicotine, provokes acute increments and accumulation of FosB in neurons of the reward pathway, chiefly in the nucleus accumbens (Nacc) (Soderstrom, Qin, Williams, Taylor, & McMillen, 2007). The FosB protein exhibits a truncated and stable splicing variant denominated deltaFosB, whose accumulation is critical for the establishment of addictive behaviors (Kowia nski et al., 2018). Cocaine induces fosb gene expression through inhibition of HDACs (Nestler, 2008). Likewise, it has been found that nicotine inhibits HDACs and produces robust H3 and H4 acetylation at the FosB promoter in the mouse striatum inducing FosB expression (Levine et al., 2011). Nevertheless, it remains to be examined whether nicotine inhibition of HDAC activity leads to a more widespread acetylation of histones at other gene’s promoters. The ability of nicotine to hyperacetylate chromatin by inhibiting HDACs could cause both transient and stable long-term acetylation states. 45.2.2.2 Effect of HDACi and Nicotine on Memory The widespread distribution throughout the nervous system of nicotinic acetylcholine receptors (nAChRs)
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may account for nicotine-induced multiple physiological effects. Several studies have demonstrated that nicotine enhances cognitive performance in smokers. It has also been repeatedly reported that nicotine is a positive regulator of learning, memory, and attention. Genetic studies have demonstrated that both dopamine receptors (DRs) and nAChRs participate in nicotine-induced cognitive improvement (Herman & Sofuoglu, 2010). Furthermore, it has been reported that nicotine regulates the synaptic mechanisms of learning that contribute to the addiction process (Subramaniyan & Dani, 2015). In an interesting work, a preexposure to nicotine effect on cocaine reward was analyzed in rodents. They found that application of the histone deacetylase inhibitor suberoylanilide hydroxamic acid mimicked the potentiating effect of nicotine on the cocaine-induced long-term potentiation in the dentate gyrus (Huang et al., 2014). These findings agreed with previous studies indicating that nicotine could have a role as an HDAC inhibitor favoring specific gene transcription (Levine et al., 2011). A few studies have demonstrated the effect of sodium butyrate (an HDAC inhibitor) on novel object recognition (NOR) memory. Long-term memory for NOR is especially sensitive to modifications in histone acetylation. The findings showed that favoring a histone hyperacetylated state via HDAC inhibition, a cognitive event that would normally be forgotten was converted into a long-term memory (Stefanko, Barrett, Ly, Reolon, & Wood, 2009). Thus, long-term memory requires structural chromatin modifications caused by histone acetylation, which provides epigenetic cues that retain gene expression associated with consolidated memories. It has been found that reducing histone acetylation, by mutating histone acetyltransferases, impaired long-term memory for object location, whereas enhancing histone acetylation, by inhibiting histone deacetylases, improved long-term object location memory. Moreover, intrahippocampal administration of the nonspecific HDAC inhibitor TSA or the class I HDAC-selective inhibitor MS275 enhanced long-term object location memory indicating the importance of inhibiting class I HDAC activity for consolidating hippocampus-dependent spatial memory (Hawk, Florian, & Abel, 2011). On the other hand, using zebra fish as an experimental model system, we evaluated memory for objects in the presence of drugs that affect attention and memory retention in rodents, such as nicotine and the HDACi PhB. An originally modified object recognition task was used to distinguish between familiar and novel objects (Faillace, Pisera-Fuster, Medrano, Bejarano, & Bernabeu, 2017). We have demonstrated for the first time that zebra fish have an innate preference for exploring some colored objects rather than others. Moreover, zebra fish were better at discriminating color changes than modest changes in shape or size. These findings also indicated that objects must be previously evaluated to
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understand zebra fish innate preference or aversion toward the object (Fig. 45.2). Moreover, nicotine significantly enhanced short-term innate novel object preference or recognition, whereas PhB showed similar effects on long-term innate object preference or recognition. The physiological consequences of HDAC inhibition by the PhB treatment can only be observed several hours after exposure to PhB since its effects are mediated by
gene transcription. In contrast, acute nicotine effects can be observed more rapidly and transiently. The findings suggested that nicotine and PhB can facilitate object recognition in zebra fish, which may involve memory. Interestingly, when one of the paired objects in the testing session was a naturally preferred object whereas the other was a nonpreferred object, nicotine and PhB generally potentiated but in some cases inverted naive preferences.
FIG. 45.2 Nicotine and phenylbutyrate effect on novel object preference for zebra fish. Zebra fish were exposed to two identical red cubes (in this case) during the training session and then either to the same red cubes (A) or to one red cube (familiar object) and one yellow ball (novel object) (B) during the testing session. Zebra fish were exposed to tank water, nicotine, phenylbutyrate (PhB, a histone deacetylase inhibitor), or phenylbutyrate + nicotine immediately after training for 10 min. Drugs were dissolved in the water tank immediately before introducing the fish. Testing sessions were performed 1.5 h (C and E) or 24 h after training (D and F). Object preference in testing sessions is depicted in the bar graphs and was assessed contrasting two red cubes (C and D) or a red cube with a yellow sphere (E and F) in the absence of the drugs. Zebra fish showed naive preference for red and aversion for yellow objects. Zebra fish can discriminate color changes and modest changes in shape of objects of the same size. Nicotine significantly enhanced short-term novel object preference or aversion (depending on the color of the object), whereas phenylbutyrate showed a similar effect at 24 h after training (hpt). Discrimination index percentage (DI) was calculated as follows: ((time of exploration of the novel object) (time of exploration of the familiar object)/(time of exploration of the novel object + time of exploration of the familiar object)) 100 (Stefanko et al., 2009). Using the DI, positive values indicate the animal spent more time exploring the novel object, whereas negative values indicate the animal preferred exploring the familiar object. The figure shows that zebra fish explored for longer periods the familiar red object than the novel yellow object when they were exposed to nicotine and tested after an interval of 1.5 h or to PhB and tested with a delay of 24 h. Data are depicted as mean SEM; n ¼ 10–12 animals per group. *P < 0.05; **P < 0.01; by Newman-Keuls test after ANOVA. “Unpublished figure and data.”
45.2 HISTONE ACETYLATION AND DEACETYLATION BY ACETYLTRANSFERASES AND DEACETYLASES
These effects do not necessarily imply memory but may involve perception and attention improvement. So, nicotine and PhB changed naive perceptual acuity and modified short- and long-term memory, respectively. 45.2.2.3 Effects of HDACi on the Reinforcing Properties of Nicotine The ability of nicotine to alter firing of dopamine neurons of the ventral tegmental area (VTA) that release DA in the NAcc is the first step leading to nicotine reward, but activation of intracellular signaling pathways downstream of nAChR is likely to be critical for the long-term consequences of nicotine exposure including conditioned reward (Walters, Cleck, Kuo, & Blendy, 2005). These studies also identified the VTA and the NAcc as the brain regions where cyclic-AMP-responsive element-binding protein (CREB) activity is essential for the establishment of nicotineinduced conditioning place preference (CPP). Our studies demonstrated that nicotine during CPP induced the phosphorylation of CREB (pCREB) and Fos expression in mesolimbic brain areas (Pascual, Pastor, & Bernabeu, 2009). The acquisition and maintenance of nicotine-induced CPP but not of nicotine-induced conditioning place aversion (CPA) increased both pCREB and Fos protein in the VTA, as well as in the NAcc, prefrontal cortex (PFC), and dorsal striatum (dStr) suggesting that nicotine associated with specific environmental cues induces changes in the neuronal activity of the different structures forming the mesolimbic pathway (Pascual et al., 2009; Walters et al., 2005). Moreover, the increase in pCREB was abolished by a treatment with mecamylamine (a nonselective nAChR antagonist) suggesting a specific activation of CREB induced by the nicotine-environment association. Histone deacetylases differentially regulate target genes of pCREB by contributing to either the activation or cessation of transcription (Fass, Butler, & Goodman, 2003). It has been described that HDAC1 can form a complex with pCREB and protein phosphatase 1 (PP1), which can cause pCREB dephosphorylation and acetyl group removal inhibiting gene transcription (Canettieri et al., 2003). In fact, the HDAC inhibitor TSA enhances activation of CRE reporter genes by cAMP. So, pCREB activity promoting specific gene transcription is at least in part inhibited by the recruitment of HDACs. The effect of HDACs on drug addiction was principally examined in experiments performed with selfadministered cocaine (Kumar et al., 2005; Romieu et al., 2008) or conditioning place preference in rats (Raybuck, McCleery, Cunningham, Wood, & Lattal, 2013). There are only two studies that assessed the effect of HDAC inhibition on the rewarding properties of nicotine in rats (Castino, Cornish, & Clemens, 2015; Pastor, Host, Zwiller, & Bernabeu, 2011). Nicotine provokes long-term modifications in brain structures, but little is known
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about the mechanism involved in nicotine-preference long-lasting neuroplasticity (Barik & Wonnacott, 2009). The epigenetic machinery is one of the main candidates to be considered for understanding nicotine biological action on the brain. As aforementioned, histone acetylation is the main modification studied so far not only for its crucial role in controlling gene expression but also because HDAC inhibitors are currently used for the treatment of cancer and neurological disorders. Regarding addiction, the effect of NaB on the extinction and reinstatement of nicotine self-administration was carefully examined. The authors found that HDAC inhibition facilitated the extinction of intravenous nicotine self-administration by enhancing extinction memory consolidation in rats (Castino et al., 2015). However, the epigenetic mechanisms involved in nicotine selfadministration and the extinction process remain unclear. In our laboratory, we used a conditioning place preference task as the alternative test to self-administration to study the rewarding properties of nicotine. By studying nicotine-induced CPP, extinction, and relapse in rats, we have demonstrated that HDAC2 protein levels increased in neurons of the reward pathway during CPP expression and relapse. In contrast, no significant change in the level of H3 acetylated at lysine 9 (H3-K9Ac) was found (Figs. 45.3 and 45.4). Furthermore, we have demonstrated that PhB dramatically reduced the preference for nicotine in a CPP task without altering the aversive properties of the drug (Pastor et al., 2011). In fact, high doses of nicotine induced place aversion (Le Foll & Goldberg, 2005; Pascual et al., 2009), which was not affected by the PhB treatment. These findings suggested that the mechanism underlying nicotine preference differs from that underlying aversion for this drug. Behavioral data obtained from experiments that assessed HDAC inhibitor effects on nicotine selfadministration and nicotine-induced CPP indicated that HDAC activity participates in appetitive drug-seeking behaviors without apparent effects on aversive behaviors. HDAC activity was relevant in at least two time windows: during the consolidation of unconditioned stimulus-conditioned stimulus association (observed by using the pavlovian CPP) and during the extinction of the conditioned memory, which was observed by using nicotine self-administration. Development and extinction of drug-cue association behaviors represent appropriate time windows for reducing the reinforcing properties of abused drugs using HDAC inhibitors. In order to characterize some mechanisms likely involved in the inhibitory effect of PhB on nicotineinduced CPP, the level of histone acetylation, the expression of HDAC2 and methyl-CpG-binding protein 2 (MeCP2), and the phosphorylation of CREB were analyzed in our laboratory. To evaluate the effect of PhB, the acetylation level of H3-K9Ac was assessed. The
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FIG. 45.3 HDAC2 and histone 3 acetylated at lysine 9 (H3K9Ac) immunoreactivity in the nucleus accumbens (Nacc) of rat. The photomicrographs show representative images of HDAC2- and H3-Ac-positive immunostaining in the Nacc, core (A) and shell (B), of different experimental groups of rats: saline (control group), CPP (nicotine-conditioned place preference group), extinction (extinction of the nicotine-induced CPP group), and relapse (reinstatement of the nicotineinduced CPP group). See Pascual et al. (2009) for details about the behavioral groups of rats. CPP, conditioning place preference. Scale bar, 50 μm. “Unpublished images.”
FIG. 45.4 Quantitative analysis of HDAC2- and H3-Ac-positive cells in different structures of the mesolimbic pathway of rat. HDAC2- (A, C) and H3-K9Ac-immunopositive cells (B, D) were found in the nucleus accumbens (NAcc, core and shell), dorsal striatum (dStr), and prelimbic frontal area (PrL) in different experimental groups of rats (see Fig. 45.3). A stringent criterion was applied to count as immunopositive cells only densities up of the mean values (darker) since every cell constitutively expresses both markers. Bars indicate mean values SEM; n ¼ 7–9 animals per group. **P < 0.01 between behavioral groups using Newman-Keuls test after ANOVA. HDAC2, histone deacetylase 2; H3-K9Ac, histone 3 acetylated at lysine 9. “Unpublished data.”
MINI-DICTIONARY OF TERMS
animals treated with PhB showed an enhancement in the acetylation level of two- to threefolds. Moreover, the number of cells expressing pCREB in different structures of the mesolimbic pathway in nicotine-conditioned rats exhibited a slight decrease caused by the HDAC inhibitor treatment. These findings suggested that whereas CREB phosphorylation is necessary to establish CPP (Pascual et al., 2009), it apparently plays no essential role in the reduction of CPP induced by the HDAC inhibitor during conditioning (Pastor et al., 2011). Interestingly, the treatment with PhB significantly reduced the number of HDAC2-immunopositive cells in the striatum of rats of the nicotine-induced CPP group, whereas this parameter remained unchanged in the striatum of animals of the nicotine-induced CPA group. Therefore, HDAC2 was required for nicotine-conditioned place preference, and it was downregulated when the drug-seeking behavior was reduced. These findings strengthened the role of HDAC2 in learning (Franklin & Mansuy, 2010). Finally, MeCP2 builds a complex with mSin3A (an essential component of the HDAC1/2 repressor complex), and HDAC2 binds to methylated DNA and strongly inhibits gene transcription (Yang & Seto, 2008). Similarly to the experiments performed with cocaine (Cassel et al., 2006), nicotine-induced CPP increases the number of cells expressing MeCP2 in the dStr without affecting the NAcc or PFC in rats (Pastor et al., 2011). Taken together, our findings indicated that HDAC inhibition was able to modulate the drug-seeking behavior associated with nicotine reward. Furthermore, HDAC2 plays a prominent role in developing the synaptic plasticity in the NAcc and dStr of the mesolimbic pathway necessary for establishing place-conditioned nicotine preference.
45.3 APPLICATIONS TO TREATMENTS Widely use and abuse of tobacco cigarettes and nicotine within them by humans are considered the principal factors causing addiction. Pharmaceutical companies and public health programs have developed and assess compounds to inhibit histone deacetylase activity (HDAC). These compounds are therapeutic tools for effectively treating different types of cancer and neuropsychiatric disorders. Furthermore, scientific reports indicate that HDAC inhibitors can control addictive behaviors such as alcohol and cocaine consumption and could be very effective for avoiding nicotine dependence and relapse. Moreover, pCREB and deltaFosB activation is responsible for nicotine reward, and these molecules may also be considered as therapeutic targets to help finishing with human tobacco use and abuse.
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MINI-DICTIONARY OF TERMS Addiction A life-lasting modification of the synaptic plasticity in the mesolimbic dopamine system in the animal’s brain, which once established induces a seeking behavior to obtain a particular reward or to avoid displeasure. The animal’s brain rewarding circuit (mesolimbic pathway) has been potentiated and controls so much the animal’s behavior that is almost exclusively oriented to seek and obtain the reward. Conditioning place preference Pavlovian or classical conditioning task that assays an animal’s free will to visit more frequently or spend more time in a place that has been previously associated with a rewarding drug. The unconditioned stimulus is a particular drug, whereas the conditioned stimulus is represented by a specific environment where the animal receives the drug. The drug-environment association can be established in a neutral environment for the animal or in a naively nonpreferred environment, which can further evidence the induction of a drug-seeking behavior provided the conditioning was established. DeltaFosB and FosB DeltaFosB and FosB are transcription factors and members of the Fos family. The fosb gene can express FosB and deltaFosB as splicing variants, which are involved in motivation and reward for abused drugs and induce the neuroadaptations that establish addictive behaviors. Addictive behaviors can be generated in mice by overexpressing deltaFosB in the striatum without exposure to drugs (McClung et al., 2004). Epigenetic Permanent or long-lasting chemical enzymatic modifications, such as methylation or acetylation of the DNA or the histones in the nucleosome (or sometimes in the cytoplasm in the case of histones) that modify chromatin structure (relaxation vs condensation) and the likelihood of specific gene expression. These modifications can be inherited but do not affect DNA nucleotide sequence or protein identity. Epigenetic mechanisms favor or repress the transcription of specific genes involved in different biological processes. Histone acetyltransferase Enzyme with a catalytic activity that deals with transferring acetyl groups mainly to lysine residues of the histones from other molecules in the cytoplasm or nucleus of the cell or in an appropriate artificial reaction media. Histone deacetylases (HDACs) Enzymes localized in the nucleus and cytoplasm of cells that regulate the degree of acetylation of histones forming the nucleosome. They catalyze the deacetylation of mainly lysine residues in histones. A high histone deacetylase activity conducts to a low degree of acetylation (a low number of acetylated lysine residues), which drives to DNA condensation inhibiting gene transcription (Fig. 45.1). Histone deacetylase 2 is the most prominent HDAC expressed in the mammalian brain. Methyl-CpG-binding protein 2 (MeCP2) This protein can repress gene transcription by specifically binding to methylated promoters on genomic regions with a high frequency of cytosines and guanines (CpG islands). MeCP2 binding to 5-methylcytosines facilitates the recruitment of chromatin remodeling and transcriptional repressor complexes, which results in a condensed chromatin state. The protein has a critical role in breast cancer progression and embryonic development. Mutations of the gene encoding this protein (MBD2) may cause Rett syndrome. It may also function as a demethylase to promote transcription. Phenylbutyrate (PhB) A chemical compound that inhibits histone deacetylase activity. PhB may increase the likelihood of gene transcription by tilting the balance toward a hyperacetylated state of histones forming the nucleosomes (Fig. 45.1). Phosphorylated cyclic AMP response element-binding transcription factor (pCREB) CREB protein is activated by phosphorylation regulated by kinases in the cytoplasm of cells that respond to different cell signaling pathways. pCREB translocates to the nucleus where it
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regulates the transcription of a battery of genes by binding to CRE sequences in gene promoter regions. pCREB enhancement suggests functional activation of a cell, such as depolarization of an excitable cell. Synaptic plasticity or neuroadaptations Any long-lasting morphological or molecular change that significantly modifies synaptic efficacy or activity in a neural circuit. Sprouting, dendritic or synaptic terminal retraction, spine formation, pruning, membrane receptor up- and downregulation, neurotransmitter release or uptake up- and downregulation, pre- and/or postsynaptic efficacy modifications such as long-term potentiation, long-term depression, and synaptic facilitation.
Key Facts • Key fact of epigenetics: The epigenetic machinery is one of the main candidates to be considered for understanding nicotine-induced long-lasting changes in synaptic plasticity in the brain. • Key fact of nicotine-environment associations and the mesolimbic pathway: Nicotine associated with specific environmental cues in a conditioning task induces changes in the neuronal activity of different structures of the mesolimbic pathway. • Key fact of histone deacetylases (HDAC): HDAC inhibition facilitates the extinction of intravenous nicotine self-administration in rats. • Key fact of histone deacetylase 2 (HDAC2): HDAC2 plays a prominent role in triggering changes in the neuronal activity of the nucleus accumbens and dorsal striatum of the mesolimbic pathway necessary for establishing nicotine-conditioned place preference. • Key fact of histone deacetylases and phosphorylated CREB: Histone deacetylases differentially regulate the target genes of phosphorylated CREB by contributing to either the activation or cessation of transcription. • Key fact of phosphorylated CREB: CREB phosphorylation is necessary to establish nicotineconditioned place preference but apparently plays no essential role in the reduction of nicotine-conditioned place preference caused by the histone deacetylase inhibitor phenylbutyrate. • Key fact of histone deacetylase inhibitors: These inhibitors are currently used for medical treatment of life-threatening diseases, and they could be used at appropriate doses to treat nicotine addiction and help smokers to quit. Summary Points • We have described herein published results about histone deacetylase inhibitor effects on animal behavior and protein expression involved in the rewarding properties of nicotine. • Histone deacetylase activity inhibition enhanced consolidation of the extinction memory and hence attenuated nicotine reinstatement.
• Histone deacetylase 2 was required for nicotineinduced place preference conditioning and was downregulated when the drug-seeking behavior was reduced. • Empirical evidences indicate that nicotine could have a role as a histone deacetylase inhibitor regulating synaptic plasticity in particular brain areas by favoring specific gene transcription. • Histone deacetylase activity inhibitors increased aversive long-term memory but reduced nicotine place preference. • Nicotine induced short-term improvement, while histone deacetylase inhibition induced long-term enhancement of object perception and memory for object recognition.
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Jiang, Y., Langley, B., Lubin, F. D., Renthal, W., Wood, M. A., Yasui, D. H., et al. (2008). Epigenetics in the nervous system. The Journal of Neuroscience, 28(46), 11753–11759. Kouzarides, T. (2007). Chromatin modifications and their function. Cell, 128(4), 693–705. Kowia nski, P., Lietzau, G., Steliga, A., Czuba, E., Ludkiewicz, B., Waskow, M., et al. (2018). Nicotine-induced CREB and DeltaFosB activity is modified by caffeine in the brain reward system of the rat. Journal of Chemical Neuroanatomy, 88, 1–12. Kumar, A., Choi, K. H., Renthal, W., Tsankova, N. M., Theobald, D. E., Truong, H. T., et al. (2005). Chromatin remodeling is a key mechanism underlying cocaine-induced plasticity in striatum. Neuron, 48(2), 303–314. Le Foll, B., & Goldberg, S. R. (2005). Nicotine induces conditioned place preferences over a large range of doses in rats. Psychopharmacology, 178(4), 481–492. Levine, A., Huang, Y., Drisaldi, B., Griffin, E.A Jr., Pollak, D.D., Xu, S., Yin, D., Schaffran, C., Kandel, D.B., & Kandel, E.R. (2011). Molecular mechanism for a gateway drug: epigenetic changes initiated by nicotine prime gene expression by cocaine. Sci Transl med, 3 (107):107ra109. Marmorstein, R., & Zhou, M. M. (2014). Writers and readers of histone acetylation: structure, mechanism, and inhibition. Cold Spring Harbor Perspectives in Biology, 6(7), a018762. McClung, C. A., Ulery, P. G., Perrotti, L. I., Zachariou, V., Berton, O., & Nestler, E. J. (2004). DeltaFosB: a molecular switch for long-term adaptation in the brain. Brain Research. Molecular Brain Research. 132, 146–154. Nestler, E. J. (2008). Transcriptional mechanisms of addiction: role of DeltaFosB. Review. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, 363(1507), 3245–3255. O’Brown, Z. K., & Greer, E. L. (2016). N6-methyladenine: a conserved and dynamic DNA mark. Advances in Experimental Medicine and Biology, 945, 213–246. Pascual, M. M., Pastor, V., & Bernabeu, R. O. (2009). Nicotineconditioned place preference induced CREB phosphorylation and Fos expression in the adult rat brain. Psychopharmacology, 207(1), 57–71. Pastor, V., Host, L., Zwiller, J., & Bernabeu, R. O. (2011). Histone deacetylase inhibition decreases preference without affecting aversion for nicotine. Journal of Neurochemistry, 116(4), 636–645.
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Peng, L., Yuan, Z., Ling, H., Fukasawa, K., Robertson, K., Olashaw, N., et al. (2011). SIRT1 deacetylates the DNA methyltransferase 1 (DNMT1) protein and alters its activities. Molecular and Cellular Biology, 31(23), 4720–4734. Raybuck, J. D., McCleery, E. J., Cunningham, C. L., Wood, M. A., & Lattal, K. M. (2013). The histone deacetylase inhibitor sodium butyrate modulates acquisition and extinction of cocaine-induced conditioned place preference. Pharmacology, Biochemistry, and Behavior, 106, 109–116. Romieu, P., Host, L., Gobaille, S., Sandner, G., Aunis, D., & Zwiller, J. (2008). Histone deacetylase inhibitors decrease cocaine but not sucrose self-administration in rats. The Journal of Neuroscience, 28(38), 9342–9348. Sakharkar, A. J., Zhang, H., Tang, L., Baxstrom, K., Shi, G., Moonat, S., et al. (2014). Effects of histone deacetylase inhibitors on amygdaloid histone acetylation and neuropeptide Y expression: a role in anxietylike and alcohol-drinking behaviours. The International Journal of Neuropsychopharmacology, 17(8), 1207–1220. Soderstrom, K., Qin, W., Williams, H., Taylor, D. A., & McMillen, B. A. (2007). Nicotine increases FosB expression within a subset of reward- and memory-related brain regions during both peri- and post-adolescence. Psychopharmacology, 191, 891–897. Stefanko, D. P., Barrett, R. M., Ly, A. R., Reolon, G. K., & Wood, M. A. (2009). Modulation of long-term memory for object recognition via HDAC inhibition. Proceedings of the National Academy of Sciences of the United States of America, 106(23), 9447–9452. Subramaniyan, M., & Dani, J. A. (2015). Dopaminergic and cholinergic learning mechanisms in nicotine addiction. Annals of the New York Academy of Sciences, 1349, 46–63. Tollefsbol, T. (2011). Handbook of epigenetics, the new molecular and medical genetics (1st ed.). London: Academic Press. Vaissière, T., Sawan, C., & Herceg, Z. (2008). Epigenetic interplay between histone modifications and DNA methylation in gene silencing. Mutation Research, 659(1–2), 40–48. Walters, C. L., Cleck, J. N., Kuo, Y. C., & Blendy, J. A. (2005). Mu-opioid receptor and CREB activation are required for nicotine reward. Neuron, 46(6), 933–943. Yang, X. J., & Seto, E. (2007). HATs and HDACs: from structure, function and regulation to novel strategies for therapy and prevention. Oncogene, 26(37), 5310–5318. Yang, X. J., & Seto, E. (2008). Lysine acetylation: codified crosstalk with other posttranslational modifications. Molecular Cell, 31(4), 449–461.
45 Effects of Nicotine and Histone Deacetylase Inhibitors on the Brain Maria Paula Faillace, Ramon O. Bernabeu Departamento de Fisiologı´a e Instituto de Fisiologı´a y Biofı´sica (IFIBIO-Houssay, UBA-CONICET), Universidad de Buenos Aires (UBA), Paraguay 2155, Buenos Aires, Argentina
Abbreviations CPA CpG CPP CREB DNMT dStr H3 H4 HAT HDAC HDACi NaB Nacc nAChR NOR OR pCREB PFC PhB PrL TSA VTA
conditioning place aversion cytosine-guanine island conditioning place preference cyclic-AMP-responsive element-binding protein DNA methyltransferase dorsal striatum histone 3 histone 4 histone acetyltransferase histone deacetylase HDAC inhibitor sodium butyrate nucleus accumbens nicotinic acetylcholine receptor novel object recognition object recognition phosphorylated CREB prefrontal cortex phenylbutyrate prelimbic frontal area trichostatin A ventral tegmental area
45.1 INTRODUCTION Genetic information is stored in extremely long linear sequences of nucleotides, so the genetic material in eukaryotes is arranged into complex structures, which efficiently pack DNA within the cell nucleus (Horn & Peterson, 2002). DNA chains together with nuclear proteins form the chromatin. The histones (H) are the chief structural and constitutive regulatory proteins in chromatin. One hundred and forty-six base pairs of DNA are wrapped twice around an octamer of histones. Each octamer consists of two subunits of each type of histone: H2A, H2B, H3, and H4. This basic structural unit of chromatin is termed nucleosome.
Neuroscience of Nicotine https://doi.org/10.1016/B978-0-12-813035-3.00045-9
Epigenesis involves effects determined by a huge array of chromatin changes, which are principally induced by DNA methylation and histone postranslational modifications ( Jiang et al., 2008). Other processes can also control gene transcription by chromatin structural remodeling, and all epigenetic changes are heritable and reversible. However, no changes in DNA or protein sequence occur during epigenetic regulation. DNA methylation is the core of long-term epigenetic changes, and for several years, DNA methylation was conceived only as cytosine methylation. However, a few years ago, it was found that guanine and adenine can be methylated (O’Brown & Greer, 2016). Furthermore, histone enzymatic modifications are the most common and studied epigenetic changes that can induce long- and short-term changes in the chromatin structure. Histones have a tail that extends out of the octamer core of the nucleosome. The amino-terminal tails undergo postranslational modifications including acetylation, methylation, phosphorylation, ADP-ribosylation, farnesylation, proline isomerization, and ubiquitination (Tollefsbol, 2011). These chemical groups modify histone-DNA and histone-protein interactions remodeling chromatin with consequences for transcription and DNA replication and repair ( Jenuwein & Allis, 2001). Hence, gene expression can be driven by the acetylation and methylation of histones, which modifies DNA-protein associations and chromatin structure (Fig. 45.1). Chromatin relaxed or condensed forms favor or reduce transcription, respectively. The acetylation and demethylation of residues in histones induce DNA relaxation and promote transcription factor and remodeling protein interaction (Kouzarides, 2007; Marmorstein & Zhou, 2014). In this chapter, we analyzed epigenetic mechanisms that involve histone acetylation. Moreover, we described published results about histone deacetylase inhibitor
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FIG. 45.1 Histone acetylation and deacetylation are epigenetic mechanisms, which provoke opposite effects on chromatin structure and gene expression. Cartoon representing the epigenetic mechanism of histone acetylation of lysine residues located at histone 3 and 4 (H3 and H4) tails. Acetylation and deacetylation of histones mediate conversion between relaxed and condensed chromatin forms, respectively. (A) Histone tails are fully acetylated that induces an open state of the chromatin, which is more accessible to transcription factor interaction promoting transcription. The acetylated state is promoted by histone acetyltransferase activity. (B) Histone tail deacetylation condenses chromatin, which reduces transcription. The removal of acetyl groups from lysine residues is achieved by histone deacetylase activity. (C) Intermediate acetylated states promote a heterogeneous chromatin structure that differentially regulates transcription. Histone deacetylase inhibitors (HDACi) induce higher degrees of acetylation generally favoring gene expression. Epigenetic regulators such as nicotine can induce acetylation of specific portions of the chromatin differentially regulating gene expression. “Unpublished and original art work by R. O. Bernabeu.”
(HDACi) effects on animal behavior and protein expression related to nicotine rewarding properties.
45.2 HISTONE ACETYLATION AND DEACETYLATION BY ACETYLTRANSFERASES AND DEACETYLASES Many factors from the environment can activate the enzymes that regulate histone modifications. Histone acetylation plays a prominent role affecting chromatin structure. Acetylation of histones is a dynamic and reversible process controlled by a family of enzymes called histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs transfer acetyl groups to lysine residues, and HDACs remove acetyl groups from these amino acids. The first HAT identified called Hatl was found in yeast in 1995, and 1 year later, the first HDAC
was purified and termed HDAC-l (Yang & Seto, 2007). HAT and HDAC activation provides a mechanism to understand how histone acetylation and transcription are regulated. For instance, studies dealing with type A HATs (GCN5, p300/CBP, TAFII250, p55, and PCAF) provided information about acetylation of nucleosome histones, whereas studies dealing with type B HATs informed about acetylation mechanisms of newly synthesized histones in the cytoplasm ( Jeanteur, 2005).
45.2.1 Histone Deacetylase Inhibitors HDAC inhibitors maintain histone acetylation and can tip the balance to an “open” configuration state of chromatin leading to a transcriptional permissive state. Different drugs characterized as HDAC inhibitors have been used for the treatment of neuropsychiatric and drug addiction diseases. For instance, valproate is an HDAC inhibitor that has been extensively used for treating
45.2 HISTONE ACETYLATION AND DEACETYLATION BY ACETYLTRANSFERASES AND DEACETYLASES
neuropsychiatric disorders. Valproate induces DNA demethylation in the adult mouse brain (Dong, Chen, Gavin, Grayson, & Guidotti, 2010). Likewise, trichostatin A (TSA) increases histone acetylation and decreases DNA methylation. However, treatments with an inhibitor of DNA methyltransferases did not affect histone acetylation suggesting that acetylation can modulate methylation but not the opposite (Vaissière, Sawan, & Herceg, 2008). SIRT1 (class III HDAC) deacetylates DNA methyltransferase 1 (DNMT1), so it is likely that HDAC activity inhibitors regulate DNA methylation levels by modifying a key methyltransferase activity (Peng et al., 2011). Furthermore, the administration of HDAC inhibitors at the appropriate time point alleviates the negative dysphoric effects observed in alcohol withdrawal (Sakharkar et al., 2014). At present, more than 40 drugs that work as HDAC inhibitors have been described. Some of them were approved by the Food and Drug Administration (FDA) and are principally used to treat different cancer types, whereas other compounds are waiting for approval for clinical use. In this chapter, we focused in two HDAC inhibitors: sodium butyrate (NaB) and phenylbutyrate (PhB), which have demonstrated significant biological and behavioral effects on animal models for the study of nicotine dependence.
45.2.2 Histone Deacetylase and Nicotine Effects on the Nervous System 45.2.2.1 Nicotine Mimics the Action of HDAC Inhibitors Consumption of abused drugs, including nicotine, provokes acute increments and accumulation of FosB in neurons of the reward pathway, chiefly in the nucleus accumbens (Nacc) (Soderstrom, Qin, Williams, Taylor, & McMillen, 2007). The FosB protein exhibits a truncated and stable splicing variant denominated deltaFosB, whose accumulation is critical for the establishment of addictive behaviors (Kowia nski et al., 2018). Cocaine induces fosb gene expression through inhibition of HDACs (Nestler, 2008). Likewise, it has been found that nicotine inhibits HDACs and produces robust H3 and H4 acetylation at the FosB promoter in the mouse striatum inducing FosB expression (Levine et al., 2011). Nevertheless, it remains to be examined whether nicotine inhibition of HDAC activity leads to a more widespread acetylation of histones at other gene’s promoters. The ability of nicotine to hyperacetylate chromatin by inhibiting HDACs could cause both transient and stable long-term acetylation states. 45.2.2.2 Effect of HDACi and Nicotine on Memory The widespread distribution throughout the nervous system of nicotinic acetylcholine receptors (nAChRs)
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may account for nicotine-induced multiple physiological effects. Several studies have demonstrated that nicotine enhances cognitive performance in smokers. It has also been repeatedly reported that nicotine is a positive regulator of learning, memory, and attention. Genetic studies have demonstrated that both dopamine receptors (DRs) and nAChRs participate in nicotine-induced cognitive improvement (Herman & Sofuoglu, 2010). Furthermore, it has been reported that nicotine regulates the synaptic mechanisms of learning that contribute to the addiction process (Subramaniyan & Dani, 2015). In an interesting work, a preexposure to nicotine effect on cocaine reward was analyzed in rodents. They found that application of the histone deacetylase inhibitor suberoylanilide hydroxamic acid mimicked the potentiating effect of nicotine on the cocaine-induced long-term potentiation in the dentate gyrus (Huang et al., 2014). These findings agreed with previous studies indicating that nicotine could have a role as an HDAC inhibitor favoring specific gene transcription (Levine et al., 2011). A few studies have demonstrated the effect of sodium butyrate (an HDAC inhibitor) on novel object recognition (NOR) memory. Long-term memory for NOR is especially sensitive to modifications in histone acetylation. The findings showed that favoring a histone hyperacetylated state via HDAC inhibition, a cognitive event that would normally be forgotten was converted into a long-term memory (Stefanko, Barrett, Ly, Reolon, & Wood, 2009). Thus, long-term memory requires structural chromatin modifications caused by histone acetylation, which provides epigenetic cues that retain gene expression associated with consolidated memories. It has been found that reducing histone acetylation, by mutating histone acetyltransferases, impaired long-term memory for object location, whereas enhancing histone acetylation, by inhibiting histone deacetylases, improved long-term object location memory. Moreover, intrahippocampal administration of the nonspecific HDAC inhibitor TSA or the class I HDAC-selective inhibitor MS275 enhanced long-term object location memory indicating the importance of inhibiting class I HDAC activity for consolidating hippocampus-dependent spatial memory (Hawk, Florian, & Abel, 2011). On the other hand, using zebra fish as an experimental model system, we evaluated memory for objects in the presence of drugs that affect attention and memory retention in rodents, such as nicotine and the HDACi PhB. An originally modified object recognition task was used to distinguish between familiar and novel objects (Faillace, Pisera-Fuster, Medrano, Bejarano, & Bernabeu, 2017). We have demonstrated for the first time that zebra fish have an innate preference for exploring some colored objects rather than others. Moreover, zebra fish were better at discriminating color changes than modest changes in shape or size. These findings also indicated that objects must be previously evaluated to
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understand zebra fish innate preference or aversion toward the object (Fig. 45.2). Moreover, nicotine significantly enhanced short-term innate novel object preference or recognition, whereas PhB showed similar effects on long-term innate object preference or recognition. The physiological consequences of HDAC inhibition by the PhB treatment can only be observed several hours after exposure to PhB since its effects are mediated by
gene transcription. In contrast, acute nicotine effects can be observed more rapidly and transiently. The findings suggested that nicotine and PhB can facilitate object recognition in zebra fish, which may involve memory. Interestingly, when one of the paired objects in the testing session was a naturally preferred object whereas the other was a nonpreferred object, nicotine and PhB generally potentiated but in some cases inverted naive preferences.
FIG. 45.2 Nicotine and phenylbutyrate effect on novel object preference for zebra fish. Zebra fish were exposed to two identical red cubes (in this case) during the training session and then either to the same red cubes (A) or to one red cube (familiar object) and one yellow ball (novel object) (B) during the testing session. Zebra fish were exposed to tank water, nicotine, phenylbutyrate (PhB, a histone deacetylase inhibitor), or phenylbutyrate + nicotine immediately after training for 10 min. Drugs were dissolved in the water tank immediately before introducing the fish. Testing sessions were performed 1.5 h (C and E) or 24 h after training (D and F). Object preference in testing sessions is depicted in the bar graphs and was assessed contrasting two red cubes (C and D) or a red cube with a yellow sphere (E and F) in the absence of the drugs. Zebra fish showed naive preference for red and aversion for yellow objects. Zebra fish can discriminate color changes and modest changes in shape of objects of the same size. Nicotine significantly enhanced short-term novel object preference or aversion (depending on the color of the object), whereas phenylbutyrate showed a similar effect at 24 h after training (hpt). Discrimination index percentage (DI) was calculated as follows: ((time of exploration of the novel object) (time of exploration of the familiar object)/(time of exploration of the novel object + time of exploration of the familiar object)) 100 (Stefanko et al., 2009). Using the DI, positive values indicate the animal spent more time exploring the novel object, whereas negative values indicate the animal preferred exploring the familiar object. The figure shows that zebra fish explored for longer periods the familiar red object than the novel yellow object when they were exposed to nicotine and tested after an interval of 1.5 h or to PhB and tested with a delay of 24 h. Data are depicted as mean SEM; n ¼ 10–12 animals per group. *P < 0.05; **P < 0.01; by Newman-Keuls test after ANOVA. “Unpublished figure and data.”
45.2 HISTONE ACETYLATION AND DEACETYLATION BY ACETYLTRANSFERASES AND DEACETYLASES
These effects do not necessarily imply memory but may involve perception and attention improvement. So, nicotine and PhB changed naive perceptual acuity and modified short- and long-term memory, respectively. 45.2.2.3 Effects of HDACi on the Reinforcing Properties of Nicotine The ability of nicotine to alter firing of dopamine neurons of the ventral tegmental area (VTA) that release DA in the NAcc is the first step leading to nicotine reward, but activation of intracellular signaling pathways downstream of nAChR is likely to be critical for the long-term consequences of nicotine exposure including conditioned reward (Walters, Cleck, Kuo, & Blendy, 2005). These studies also identified the VTA and the NAcc as the brain regions where cyclic-AMP-responsive element-binding protein (CREB) activity is essential for the establishment of nicotineinduced conditioning place preference (CPP). Our studies demonstrated that nicotine during CPP induced the phosphorylation of CREB (pCREB) and Fos expression in mesolimbic brain areas (Pascual, Pastor, & Bernabeu, 2009). The acquisition and maintenance of nicotine-induced CPP but not of nicotine-induced conditioning place aversion (CPA) increased both pCREB and Fos protein in the VTA, as well as in the NAcc, prefrontal cortex (PFC), and dorsal striatum (dStr) suggesting that nicotine associated with specific environmental cues induces changes in the neuronal activity of the different structures forming the mesolimbic pathway (Pascual et al., 2009; Walters et al., 2005). Moreover, the increase in pCREB was abolished by a treatment with mecamylamine (a nonselective nAChR antagonist) suggesting a specific activation of CREB induced by the nicotine-environment association. Histone deacetylases differentially regulate target genes of pCREB by contributing to either the activation or cessation of transcription (Fass, Butler, & Goodman, 2003). It has been described that HDAC1 can form a complex with pCREB and protein phosphatase 1 (PP1), which can cause pCREB dephosphorylation and acetyl group removal inhibiting gene transcription (Canettieri et al., 2003). In fact, the HDAC inhibitor TSA enhances activation of CRE reporter genes by cAMP. So, pCREB activity promoting specific gene transcription is at least in part inhibited by the recruitment of HDACs. The effect of HDACs on drug addiction was principally examined in experiments performed with selfadministered cocaine (Kumar et al., 2005; Romieu et al., 2008) or conditioning place preference in rats (Raybuck, McCleery, Cunningham, Wood, & Lattal, 2013). There are only two studies that assessed the effect of HDAC inhibition on the rewarding properties of nicotine in rats (Castino, Cornish, & Clemens, 2015; Pastor, Host, Zwiller, & Bernabeu, 2011). Nicotine provokes long-term modifications in brain structures, but little is known
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about the mechanism involved in nicotine-preference long-lasting neuroplasticity (Barik & Wonnacott, 2009). The epigenetic machinery is one of the main candidates to be considered for understanding nicotine biological action on the brain. As aforementioned, histone acetylation is the main modification studied so far not only for its crucial role in controlling gene expression but also because HDAC inhibitors are currently used for the treatment of cancer and neurological disorders. Regarding addiction, the effect of NaB on the extinction and reinstatement of nicotine self-administration was carefully examined. The authors found that HDAC inhibition facilitated the extinction of intravenous nicotine self-administration by enhancing extinction memory consolidation in rats (Castino et al., 2015). However, the epigenetic mechanisms involved in nicotine selfadministration and the extinction process remain unclear. In our laboratory, we used a conditioning place preference task as the alternative test to self-administration to study the rewarding properties of nicotine. By studying nicotine-induced CPP, extinction, and relapse in rats, we have demonstrated that HDAC2 protein levels increased in neurons of the reward pathway during CPP expression and relapse. In contrast, no significant change in the level of H3 acetylated at lysine 9 (H3-K9Ac) was found (Figs. 45.3 and 45.4). Furthermore, we have demonstrated that PhB dramatically reduced the preference for nicotine in a CPP task without altering the aversive properties of the drug (Pastor et al., 2011). In fact, high doses of nicotine induced place aversion (Le Foll & Goldberg, 2005; Pascual et al., 2009), which was not affected by the PhB treatment. These findings suggested that the mechanism underlying nicotine preference differs from that underlying aversion for this drug. Behavioral data obtained from experiments that assessed HDAC inhibitor effects on nicotine selfadministration and nicotine-induced CPP indicated that HDAC activity participates in appetitive drug-seeking behaviors without apparent effects on aversive behaviors. HDAC activity was relevant in at least two time windows: during the consolidation of unconditioned stimulus-conditioned stimulus association (observed by using the pavlovian CPP) and during the extinction of the conditioned memory, which was observed by using nicotine self-administration. Development and extinction of drug-cue association behaviors represent appropriate time windows for reducing the reinforcing properties of abused drugs using HDAC inhibitors. In order to characterize some mechanisms likely involved in the inhibitory effect of PhB on nicotineinduced CPP, the level of histone acetylation, the expression of HDAC2 and methyl-CpG-binding protein 2 (MeCP2), and the phosphorylation of CREB were analyzed in our laboratory. To evaluate the effect of PhB, the acetylation level of H3-K9Ac was assessed. The
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FIG. 45.3 HDAC2 and histone 3 acetylated at lysine 9 (H3K9Ac) immunoreactivity in the nucleus accumbens (Nacc) of rat. The photomicrographs show representative images of HDAC2- and H3-Ac-positive immunostaining in the Nacc, core (A) and shell (B), of different experimental groups of rats: saline (control group), CPP (nicotine-conditioned place preference group), extinction (extinction of the nicotine-induced CPP group), and relapse (reinstatement of the nicotineinduced CPP group). See Pascual et al. (2009) for details about the behavioral groups of rats. CPP, conditioning place preference. Scale bar, 50 μm. “Unpublished images.”
FIG. 45.4 Quantitative analysis of HDAC2- and H3-Ac-positive cells in different structures of the mesolimbic pathway of rat. HDAC2- (A, C) and H3-K9Ac-immunopositive cells (B, D) were found in the nucleus accumbens (NAcc, core and shell), dorsal striatum (dStr), and prelimbic frontal area (PrL) in different experimental groups of rats (see Fig. 45.3). A stringent criterion was applied to count as immunopositive cells only densities up of the mean values (darker) since every cell constitutively expresses both markers. Bars indicate mean values SEM; n ¼ 7–9 animals per group. **P < 0.01 between behavioral groups using Newman-Keuls test after ANOVA. HDAC2, histone deacetylase 2; H3-K9Ac, histone 3 acetylated at lysine 9. “Unpublished data.”
MINI-DICTIONARY OF TERMS
animals treated with PhB showed an enhancement in the acetylation level of two- to threefolds. Moreover, the number of cells expressing pCREB in different structures of the mesolimbic pathway in nicotine-conditioned rats exhibited a slight decrease caused by the HDAC inhibitor treatment. These findings suggested that whereas CREB phosphorylation is necessary to establish CPP (Pascual et al., 2009), it apparently plays no essential role in the reduction of CPP induced by the HDAC inhibitor during conditioning (Pastor et al., 2011). Interestingly, the treatment with PhB significantly reduced the number of HDAC2-immunopositive cells in the striatum of rats of the nicotine-induced CPP group, whereas this parameter remained unchanged in the striatum of animals of the nicotine-induced CPA group. Therefore, HDAC2 was required for nicotine-conditioned place preference, and it was downregulated when the drug-seeking behavior was reduced. These findings strengthened the role of HDAC2 in learning (Franklin & Mansuy, 2010). Finally, MeCP2 builds a complex with mSin3A (an essential component of the HDAC1/2 repressor complex), and HDAC2 binds to methylated DNA and strongly inhibits gene transcription (Yang & Seto, 2008). Similarly to the experiments performed with cocaine (Cassel et al., 2006), nicotine-induced CPP increases the number of cells expressing MeCP2 in the dStr without affecting the NAcc or PFC in rats (Pastor et al., 2011). Taken together, our findings indicated that HDAC inhibition was able to modulate the drug-seeking behavior associated with nicotine reward. Furthermore, HDAC2 plays a prominent role in developing the synaptic plasticity in the NAcc and dStr of the mesolimbic pathway necessary for establishing place-conditioned nicotine preference.
45.3 APPLICATIONS TO TREATMENTS Widely use and abuse of tobacco cigarettes and nicotine within them by humans are considered the principal factors causing addiction. Pharmaceutical companies and public health programs have developed and assess compounds to inhibit histone deacetylase activity (HDAC). These compounds are therapeutic tools for effectively treating different types of cancer and neuropsychiatric disorders. Furthermore, scientific reports indicate that HDAC inhibitors can control addictive behaviors such as alcohol and cocaine consumption and could be very effective for avoiding nicotine dependence and relapse. Moreover, pCREB and deltaFosB activation is responsible for nicotine reward, and these molecules may also be considered as therapeutic targets to help finishing with human tobacco use and abuse.
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MINI-DICTIONARY OF TERMS Addiction A life-lasting modification of the synaptic plasticity in the mesolimbic dopamine system in the animal’s brain, which once established induces a seeking behavior to obtain a particular reward or to avoid displeasure. The animal’s brain rewarding circuit (mesolimbic pathway) has been potentiated and controls so much the animal’s behavior that is almost exclusively oriented to seek and obtain the reward. Conditioning place preference Pavlovian or classical conditioning task that assays an animal’s free will to visit more frequently or spend more time in a place that has been previously associated with a rewarding drug. The unconditioned stimulus is a particular drug, whereas the conditioned stimulus is represented by a specific environment where the animal receives the drug. The drug-environment association can be established in a neutral environment for the animal or in a naively nonpreferred environment, which can further evidence the induction of a drug-seeking behavior provided the conditioning was established. DeltaFosB and FosB DeltaFosB and FosB are transcription factors and members of the Fos family. The fosb gene can express FosB and deltaFosB as splicing variants, which are involved in motivation and reward for abused drugs and induce the neuroadaptations that establish addictive behaviors. Addictive behaviors can be generated in mice by overexpressing deltaFosB in the striatum without exposure to drugs (McClung et al., 2004). Epigenetic Permanent or long-lasting chemical enzymatic modifications, such as methylation or acetylation of the DNA or the histones in the nucleosome (or sometimes in the cytoplasm in the case of histones) that modify chromatin structure (relaxation vs condensation) and the likelihood of specific gene expression. These modifications can be inherited but do not affect DNA nucleotide sequence or protein identity. Epigenetic mechanisms favor or repress the transcription of specific genes involved in different biological processes. Histone acetyltransferase Enzyme with a catalytic activity that deals with transferring acetyl groups mainly to lysine residues of the histones from other molecules in the cytoplasm or nucleus of the cell or in an appropriate artificial reaction media. Histone deacetylases (HDACs) Enzymes localized in the nucleus and cytoplasm of cells that regulate the degree of acetylation of histones forming the nucleosome. They catalyze the deacetylation of mainly lysine residues in histones. A high histone deacetylase activity conducts to a low degree of acetylation (a low number of acetylated lysine residues), which drives to DNA condensation inhibiting gene transcription (Fig. 45.1). Histone deacetylase 2 is the most prominent HDAC expressed in the mammalian brain. Methyl-CpG-binding protein 2 (MeCP2) This protein can repress gene transcription by specifically binding to methylated promoters on genomic regions with a high frequency of cytosines and guanines (CpG islands). MeCP2 binding to 5-methylcytosines facilitates the recruitment of chromatin remodeling and transcriptional repressor complexes, which results in a condensed chromatin state. The protein has a critical role in breast cancer progression and embryonic development. Mutations of the gene encoding this protein (MBD2) may cause Rett syndrome. It may also function as a demethylase to promote transcription. Phenylbutyrate (PhB) A chemical compound that inhibits histone deacetylase activity. PhB may increase the likelihood of gene transcription by tilting the balance toward a hyperacetylated state of histones forming the nucleosomes (Fig. 45.1). Phosphorylated cyclic AMP response element-binding transcription factor (pCREB) CREB protein is activated by phosphorylation regulated by kinases in the cytoplasm of cells that respond to different cell signaling pathways. pCREB translocates to the nucleus where it
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45. EFFECTS OF NICOTINE AND HISTONE DEACETYLASE INHIBITORS ON THE BRAIN
regulates the transcription of a battery of genes by binding to CRE sequences in gene promoter regions. pCREB enhancement suggests functional activation of a cell, such as depolarization of an excitable cell. Synaptic plasticity or neuroadaptations Any long-lasting morphological or molecular change that significantly modifies synaptic efficacy or activity in a neural circuit. Sprouting, dendritic or synaptic terminal retraction, spine formation, pruning, membrane receptor up- and downregulation, neurotransmitter release or uptake up- and downregulation, pre- and/or postsynaptic efficacy modifications such as long-term potentiation, long-term depression, and synaptic facilitation.
Key Facts • Key fact of epigenetics: The epigenetic machinery is one of the main candidates to be considered for understanding nicotine-induced long-lasting changes in synaptic plasticity in the brain. • Key fact of nicotine-environment associations and the mesolimbic pathway: Nicotine associated with specific environmental cues in a conditioning task induces changes in the neuronal activity of different structures of the mesolimbic pathway. • Key fact of histone deacetylases (HDAC): HDAC inhibition facilitates the extinction of intravenous nicotine self-administration in rats. • Key fact of histone deacetylase 2 (HDAC2): HDAC2 plays a prominent role in triggering changes in the neuronal activity of the nucleus accumbens and dorsal striatum of the mesolimbic pathway necessary for establishing nicotine-conditioned place preference. • Key fact of histone deacetylases and phosphorylated CREB: Histone deacetylases differentially regulate the target genes of phosphorylated CREB by contributing to either the activation or cessation of transcription. • Key fact of phosphorylated CREB: CREB phosphorylation is necessary to establish nicotineconditioned place preference but apparently plays no essential role in the reduction of nicotine-conditioned place preference caused by the histone deacetylase inhibitor phenylbutyrate. • Key fact of histone deacetylase inhibitors: These inhibitors are currently used for medical treatment of life-threatening diseases, and they could be used at appropriate doses to treat nicotine addiction and help smokers to quit. Summary Points • We have described herein published results about histone deacetylase inhibitor effects on animal behavior and protein expression involved in the rewarding properties of nicotine. • Histone deacetylase activity inhibition enhanced consolidation of the extinction memory and hence attenuated nicotine reinstatement.
• Histone deacetylase 2 was required for nicotineinduced place preference conditioning and was downregulated when the drug-seeking behavior was reduced. • Empirical evidences indicate that nicotine could have a role as a histone deacetylase inhibitor regulating synaptic plasticity in particular brain areas by favoring specific gene transcription. • Histone deacetylase activity inhibitors increased aversive long-term memory but reduced nicotine place preference. • Nicotine induced short-term improvement, while histone deacetylase inhibition induced long-term enhancement of object perception and memory for object recognition.
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