Transcriptional mechanisms of drug addiction (2012)

INTRODUCTION

The study of transcriptional mechanisms of addiction is based on the hypothesis that regulation of gene expression is one important mechanism by which chronic exposure to a drug of abuse causes long-lasting changes in the brain that underlie the behavioral abnormalities that define a state of addiction.1,2) A corollary of this hypothesis is that changes induced in the functioning of several neurotransmitter systems, and in the morphology of certain neuronal cell types in the brain, by chronic drug administration are mediated in part via changes in gene expression.

Of course, not all drug-induced neural and behavioral plasticity is mediated at the level of gene expression, as we know the crucial contributions of translational and posttranslational modifications and protein trafficking in addiction-related phenomena. On the other hand, regulation of gene expression is one central mechanism and likely to be particularly crucial for the life-long abnormalities that characterize addiction. Indeed, transcriptional regulation provides a template on top of which these other mechanisms operate.

Work over the past ~15 years has provided increasing evidence for a role of gene expression in drug addiction, as several transcription factors – proteins that bind to specific responses elements in the promoter regions of target genes and regulate those genes’ expression – have been implicated in drug action. According to this scheme, shown in Fig. 1, drugs of abuse, via their initial actions at the synapse, produce changes within neurons that signal to the nucleus and regulate the activity of numerous transcription factors and many other types of transcriptional regulatory proteins.3) A These nuclear changes gradually and progressively build with repeated drug exposure and underlie stable changes in the expression of specific target genes which, in turn, contribute to the lasting changes in neural function that maintain a state of addiction.1,4)

This review focuses on several transcription factors, which have been shown to play important roles in addiction. We focus further on drug-regulated transcription factors within the brain’s reward circuitry, areas of brain that normally regulate an individual’s responses to natural rewards (e.g., food, sex, social interaction), but are corrupted by chronic drug exposure to cause addiction. This brain reward circuitry includes dopaminergic neurons in the ventral tegmental area of the midbrain and the several regions of limbic forebrain they innervate, including nucleus accumbens (ventral striatum), prefrontal cortex, amygdala, and hippocampus, among others. As will be seen, the vast majority of research on transcriptional mechanisms of addiction to date has concentrated on the nucleus accumbens.

ΔFosB

ΔFosB is encoded by the FosB gene and shares homology with other Fos family transcription factors, which include c-Fos, FosB, Fra1, and Fra2.5) These Fos family proteins heterodimerize with Jun family proteins (c-Jun, JunB, or JunD) to form active activator protein-1 (AP1) transcription factors that bind to AP1 sites present in the promoters of certain genes to regulate their transcription. These Fos family proteins are induced rapidly and transiently in specific brain regions after acute administration of many drugs of abuse (Fig. 2).2) These responses are seen most prominently in nucleus accumbens and dorsal striatum, but also seen in several other brain areas.6) All of these Fos family proteins, however, are highly unstable and return to basal levels within hours of drug administration.

Very different responses are seen after chronic administration of drugs of abuse (Fig. 2). Biochemically modified isoforms of ΔFosB (M_r 35-37 kD) accumulate within the same brain regions after repeated drug exposure, whereas all Fos family members show tolerance (that is, reduced induction compared with initial drug exposures).7–9) Such accumulation of ΔFosB has been observed for virtually all drugs of abuse, although different drugs differ somewhat in the relative degree of induction seen in nucleus accumbens core vs. shell, dorsal striatum, and other brain regions.2,6) At least for some drugs of abuse, induction of ΔFosB appears selective for the dynorphin-containing subset of medium spiny neurons – those that predominantly express D1 dopamine receptors – within striatal regions. The 35-37 kD isoforms of ΔFosB dimerize predominantly with JunD to form an active and long-lasting AP-1 complex within these brain regions,7,10) although there is some evidence from in vitro studies that ΔFosB may form homodimers.11) Drug induction of ΔFosB in the nucleus accumbens seems to be a response to the pharmacological properties of the drug per se and not related to volitional drug intake, since animals that self-administer cocaine or receive yoked drug injections show equivalent induction of this transcription factor in this brain region.6) In contrast, ΔFosB induction in certain other regions, for example, orbitofrontal cortex, requires volitional drug administration.12)

The 35-37 kD ΔFosB isoforms accumulate with chronic drug exposure due to their extraordinarily long half-lives.7–13) As a result of its stability, the ΔFosB protein persists in neurons for at least several weeks after cessation of drug exposure. We now know that this stability is due to two factors: 1) the absence in ΔFosB of two degron domains, which are present at the C-terminus of full length FosB and all other Fos family proteins and target those proteins to rapid degradation, and 2) the phosphorylation of ΔFosB at its N-terminus by casein kinase 2 and perhaps other protein kinases.14–16) The stability of the ΔFosB isoforms provides a novel molecular mechanism by which drug-induced changes in gene expression can persist despite relatively long periods of drug withdrawal. We have, therefore, proposed that ΔFosB functions as a sustained “molecular switch” that helps initiate and then maintain an addicted state.1,2)

CREB

Cyclic AMP response element binding protein (CREB) is one of the most studied transcription factors in neuroscience and has been implicated in diverse aspects of neural plasticity.33) It forms homodimers that can bind to genes at cyclic AMP response elements (CREs), but primarily activates transcription after it has been phosphorylated at Ser133 (by any of several protein kinases), which allows recruitment of CREB-binding protein (CBP) that then promotes transcription. The mechanism by which CREB activation represses the expression of certain genes is less well understood.

Both psychostimulants (cocaine and amphetamine) and opiates increase CREB activity, acutely and chronically – as measured by increased phospho-CREB (pCREB) or reporter gene activity in CRE-LacZ transgenic mice – in multiple brain regions, including the nucleus accumbens and dorsal striatum.34–36) The time course of this activation is very different from that exhibited by ΔFosB. As depicted in Fig. 2, CREB activation is highly transient in response to acute drug administration and reverts to normal levels within a day or two after withdrawal. In addition, CREB activation occurs in both the dynorphin and enkephalin subtypes of medium spiny neurons.34) In contrast to cocaine and opiates, CREB shows more complicated and varied responses to other drugs of abuse.4)

Experiments involving the inducible overexpression of CREB or a dominant negative mutant in bitransgenic mice or with viral vectors have shown that activation of CREB – in striking contrast to ΔFosB – in the nucleus accumbens decreases the rewarding effects of cocaine and of opiates as assessed in place conditioning assays.37,38) Nevertheless, CREB activation, like ΔFosB induction, promotes drug self-administration.39) Importantly, effects with dominant negative CREB have been validated with inducible knockdowns of endogenous CREB activity.39–41) It is interesting that both transcription factors drive volitional drug intake; presumably ΔFosB does so via positive reinforcement, whereas CREB induces this phenotype via negative reinforcement. The latter possibility is consistent with considerable evidence that CREB activity in this brain region causes a negative emotional state.34,42)

CREB activity has been directly linked to the functional activity of nucleus accumbens medium spiny neurons. CREB overexpression increases, whereas dominant-negative CREB decreases, the electrical excitability of medium spiny neurons.43) Possible differences between dynorphin and enkephalin neurons have not yet been explored. The observation that viral-mediated overexpression of a K⁺ channel subunit in the nucleus accumbens, which decreases medium spiny neuron excitability, enhances locomotor responses to cocaine suggests that CREB acts as a break on behavioral sensitization to cocaine by upregulating neuron excitability.43)

Drugs of abuse activate CREB in several brain regions beyond the nucleus accumbens. One example is the ventral tegmental area, where chronic administration of cocaine or opiates activates CREB within dopaminergic and non-dopaminergic neurons. This effect seems to promote or attenuate the rewarding responses of drugs of abuse depending on the subregion of the ventral tegmental area affected.

Numerous target genes for CREB have been identified, through both open-ended and candidate gene approaches, which mediate these and other effects on nucleus accumbens medium spiny neurons and the resulting CREB behavioral phenotype.18,32,36) Prominent examples include the opioid peptide dynorphin,37) which feeds back and suppresses dopaminergic signaling to the nucleus accumbens as stated earlier.24,25) Also implicated are certain glutamate receptor subunits, such as the GluA1 AMPA subunit and GluN2B NMDA subunit, as well as K⁺ and Na⁺ ion channel subunits, which together would be expected to control nucleus accumbens cell excitability.43,44) BDNF is still another target gene for CREB in nucleus accumbens, and it too is implicated in mediating the CREB behavioral phenotype.35) CREB induction also has been shown to contribute to cocaine’s induction of dendritic spines on nucleus accumbens medium spiny neurons.45)

CREB is just one of several related proteins that bind CREs and regulate transcription of target genes. Several products of the cyclic AMP response element modulator (CREM) gene regulate CRE-mediated transcription. Some of the products (e.g., CREM) are transcriptional activators, whereas others (e.g., ICER or inducible cyclic AMP repressor) function as endogenous dominant negative antagonists. In addition, several activating transcription factors (ATFs) can influence gene expression in part by binding to CRE sites. Recent studies have implicated these various transcription factors in drug responses. Amphetamine induces ICER expression in nucleus accumbens, and overexpression of ICER in this region, by use of viral-mediated gene transfer, increases an animal’s sensitivity to the behavioral effects of the drug.46) This is consistent with findings, cited above, that local overexpression of dominant negative CREB mutants or local knockdown of CREB exerts similar effects. Amphetamine also induces ATF2, ATF3, and ATF4 in nucleus accumbens, while no effect is seen for ATF1 or CREM.47) ATF2 overexpression in this region, like that of ICER, increases behavioral responses to amphetamine, while ATF3 or ATF4 overexpression has the opposite effect. Very little is known about the target genes for these various CREB family proteins, an important direction for future research.

NFκB

Nuclear factor-κB (NFκB), a transcription factor that is rapidly activated by diverse stimuli, is best studied for its role in inflammation and immune responses. It has more recently been shown to be important in synaptic plasticity and memory.48) NFκB is induced in the nucleus accumbens by repeated cocaine administration,49,50) where it is required for cocaine’s induction of dendritic spines of nucleus accumbens medium spiny neurons. Such induction of NFκB contributes to sensitization to the rewarding effects of the drug.50) A major goal of current research is to identify the target genes through which NFκB causes this cellular and behavioral plasticity.

Interestingly, cocaine induction of NFκB is mediated via ΔFosB : ΔFosB overexpression in nucleus accumbens induces NFκB, while overexpression of the ΔcJun dominant negative blocks cocaine induction of the transcription factor.21,49) Regulation of NFκB by ΔFosB illustrates the complex transcriptional cascades involved in drug action. As well, NFκB has been implicated in some of the neurotoxic effects of methamphetamine in striatal regions.51) The role of NFκB in medium spiny neuron spinogenesis has recently been extended to stress and depression models,52) a finding of particular importance considering the comorbidity of depression and addiction, and the well-studied phenomenon of stress-induced relapse to drug abuse.

MEF2

Myocyte enhancing factor-2 (MEF2) was discovered for its role in controlling cardiac myogenesis. More rerecently, MEF2 has been implicated in brain function.53) Multiple MEF2 isoforms are expressed in brain, including in nucleus accumbens medium spiny neurons, where they form homo- and heterodimers that can activate or repress gene transcription depending on the nature of the proteins they recruit. Recent work outlines a possible mechanism by which chronic cocaine suppresses MEF2 activity in the nucleus accumbens in part through a D1 receptor-cAMP-dependent inhibition of calcineurin, a Ca2⁺-dependent protein phosphatase.28) Cocaine regulation of Cdk5, which is also a target for cocaine and ΔFosB as stated earlier, may be involved as well. This reduction in MEF2 activity is required for cocaine induction of dendritic spines on medium spiny neurons. An important focus of current work is to identify the target genes through MEF2 produces this effect.

FUTURE DIRECTIONS

The transcription factors discussed above are just a few of many that have been studied over the years in addiction models. Others implicated in addiction include the glucocorticoid receptor, nucleus accumbens 1 transcription factor (NAC1), early growth response factors (EGRs), and signal transducers and activators of transcription (STATs).1,2) As just one example, the glucocorticoid receptor is required in dopaminoceptive neurons for cocaine seeking.54) The goal of future research is to obtain a more complete view of the transcription factors induced in nucleus accumbens and other brain reward regions in response to chronic exposure to drugs of abuse and to define the range of target genes they influence to contribute to the behavioral phenotype of addiction.

The other major goal of future research is to delineate the precise molecular steps by which these various transcription factors regulate their target genes. Thus, we now know that transcription factors control gene expression by recruiting to their target genes a series of co-activator or co-repressor proteins which together regulate the structure of chromatin around the genes and the subsequent recruitment of the RNA polymerase II complex which catalyzes transcription.4) For example, recent research has demonstrated that the ability of ΔFosB to induce the cdk5 gene occurs in concert with the recruitment of a histone acetyltransferase and related chromatin remodeling proteins to the gene.55) In contrast, the ability of ΔFosB to repress the c-Fos gene occurs in concert with the recruitment of a histone deacetylase and presumably several other repressive proteins such as a repressive histone methyltransferase (Fig. 3).2,9,31) Given that hundreds of chromatin regulatory proteins are likely recruited to a gene in concert with its activation or repression, this work is just the tip of the iceberg of vast amounts of information that need to be discovered in the years ahead.

    

Fig. 3

Epigenetic mechanisms of ΔFosB action. The figure illustrates the very different consequences when ΔFosB binds to a gene that it activates (e.g., Cdk5) versus represses (e.g., c-Fos). At the Cdk5 promoter (A), ΔFosB recruits histone …

As progress is made in identifying target genes for drug-regulated transcription factors, this information will provide an increasingly complete template that can be used to guide drug discovery efforts. It is hoped that new medication treatments will be developed based on these dramatic advances in our understanding of transcription mechanisms that underlie addiction.

References