Cocaine increases phosphorylation of MeCP2 in the rat striatum in vivo: A differential role of NMDA receptors

Abstract

Methyl CpG-binding protein-2 (MeCP2) is a transcriptional regulator that binds to methylated DNA at CpG sites and functions to silence DNA transcription. MeCP2 is subject to the phosphorylation modification at serine 421 (S421), which releases MeCP2 from DNA and thus facilitates gene expression. As a transcriptional repressor densely expressed in limbic reward circuits of adult mammalian brains, MeCP2 is recently emerging as a critical epigenetic factor in experience-dependent neural plasticity and psychostimulant addiction. In this study, we investigated the regulation of MeCP2 phosphorylation in the rat striatum by the psychostimulant cocaine in vivo. We found that acute systemic injection of cocaine increased MeCP2 phosphorylation at S421 in the rat striatum, including both the caudate putamen and the nucleus accumbens, while cocaine did not affect MeCP2 phosphorylation in the medial prefrontal cortex. The cocaine-stimulated MeCP2 phosphorylation in the nucleus accumbens was a rapid and transient event, as it was evident at 20 min and returned to normal levels 3 h after drug injection. The cocaine effect in the caudate putamen was however relatively delayed. Reliable induction of MeCP2 phosphorylation in this region was detected at 60 min. Pretreatment with an N-methyl-d-aspartate (NMDA) glutamate receptor antagonist significantly reduced the cocaine-stimulated MeCP2 phosphorylation in the caudate putamen, although not in the nucleus accumbens. Our data support that MeCP2 is a sensitive target of psychostimulants. Its phosphorylation status is regulated by psychostimulant exposure. NMDA receptors play a region-specific role in linking cocaine to MeCP2 phosphorylation in striatal neurons in vivo.

Introduction

Methyl CpG-binding protein-2 (MeCP2) is a founding member of the DNA-binding protein family that binds to methylated DNA at CpG sites (Lewis et al., 1992, Klose and Bird, 2006). Once bound, MeCP2 recruits a histone deacetylase (HDAC) to silence transcription of target genes. As a global transcription repressor, MeCP2 is important for neural development. Mutations of the gene (Mecp2) encoding MeCP2 cause Rett syndrome, an X-linked postnatal neurodevelopmental disorder characterized by autism-like symptoms and mental retardation (Gemelli et al., 2006, Chahrour and Zoghbi, 2007). Additionally, MeCP2 is densely expressed in postmitotic neurons of adult brains and is considered to be an epigenetic factor critical for the regulation of adult brain function. Indeed, MeCP2 is sensitive to changing neuronal activity and Ca²⁺ signals, and functions to link these changes to altered expression of various genes. For example, the neuronal activity-dependent Ca²⁺ influx induced the de novo phosphorylation of MeCP2 at serine 421 (S421) through a CaMKII-dependent mechanism. This phosphorylation contributes to releasing MeCP2 from brain-derived neurotrophic factor (BDNF) promoter III, thereby facilitating BDNF transcription, dendritic growth, and spine maturation (Chen et al., 2003, Martinowic et al., 2003, Zhou et al., 2006). Other studies also support the role of MeCP2 in the regulation of excitatory transmission and learning and memory (Moretti et al., 2006, Nelson et al., 2006, Nelson et al., 2008, Chao et al., 2007, Dani and Nelson, 2009, Gambino et al., 2010). Apparently, the MeCP2-mediated stimulus-transcription coupling is an important pathway linking neuronal activity to a program of gene expression. Aberration of this coupling may lead to various neuropsychiatric disorders (Monteggia and Kavalali, 2009).

Psychostimulant drugs of abuse (cocaine and amphetamines) have long been appreciated to induce distinct sets of gene expression in the forebrain reward circuits, including the striatum. Altered gene expression initiates and directs a series of neuroadaptations at cellular and synaptic levels, which contributes to enduring drug-seeking behavior (Chao and Nestler, 2004, McClung and Nestler, 2008). While underlying mechanisms for drug-stimulated gene expression are not fully understood, MeCP2 may play a significant role at the transcriptional level based on several recent studies (Feng and Nestler, 2010). Deng et al. (2010) reported that acute amphetamine increased MeCP2 phosphorylation at S421 in the mouse ventral striatum/nucleus accumbens (NAc). The increased MeCP2 phosphorylation correlated with behavioral sensitization to chronic amphetamine. Mecp2 hypomorphic mutant mice showed deficit of amphetamine-stimulated structural plasticity and immediate early gene expression in the NAc. Im et al. (2010) found that extended self-administration of cocaine elevated MeCP2 expression in the rat dorsal striatum/caudate putamen (CPu), similar to the early results seen after repeated cocaine injections in rats (Cassel et al., 2006). Knockdown of MeCP2 expression in the CPu through local lentiviral delivery of a short hairpin interfering RNA reversed cocaine intake (Im et al., 2010). These results indicate that MeCP2 is a sensitive target of psychostimulants. Its responses to psychostimulant exposure constitute as an essential element of overall neuroadaptations related to the persistent properties of drugs of abuse.

Since MeCP2 expression and phosphorylation at S421 in response to acute cocaine administration have been less thoroughly investigated and characterized, we carried out this study to define them in rats in vivo. We examined the effect of a single and behaviorally active dose of cocaine on total and S421-phosphorylated MeCP2 expression in the three forebrain regions: the CPu, NAc, and medial prefrontal cortex (mPFC). We then defined the time course of changed MeCP2 phosphorylation after cocaine. Finally, we evaluated the role of N-methyl-d-aspartate (NMDA) glutamate receptors in mediating the cocaine effect on MeCP2 expression and phosphorylation. In most experiments, a well-known transcriptional factor, CREB (cAMP response element-binding), was studied as a positive control.