Methamphetamine produces increased neuropeptide gene expression by TET-dependent DNA methylation/hydroxymethylation events in the rat nucleus accumbens

SUBRAMANIAM JAYANTHI; BETINA GONZALEZ; PAWARIS WONGPRAYOON; MICHAEL T. MCCOY; BRUCE LADENHEIM; JEAN LUD CADET

Washinton DC

Congreso; Neuroscience 2014, SfN's 44rd annual meeting; 2014

Society for Neuroscience

Methamphetamine (METH) addiction is a relapsing neuropsychiatric disorder that is secondary, in part, to neuroadaptations within neurocircuitries that mediate stress responses. Our recent microarray and quantitative PCR (qPCR) analyses had revealed that an injection of METH (10mg/kg) upregulated several stress-related gene markers measured one month later using tissues from the rat nucleus accumbens. These genes included corticotropin-releasing hormone (Crh/ Crf), vasopressin (Avp) and cocaine-amphetamine regulated transcription factor (Cart). The long-lasting increases in Crh, Cart, and Avp mRNA expression had suggested that METH exposure might produce prolonged activation of the endogenous stress system via epigenetic modifications that have been identified as important mediators of long-lasting memories. For example, methylation of cytosine (5mC) bases in DNA is known to cause gene silencing whereas reversible conversion of 5mC to its hydroxylated form, 5hmC, by the Ten-eleven translocation (Tet) family of enzymes can increase gene transcription. To assess if METH treatment could cause changes in DNA methylation, we used methylated DNA immunoprecipitation (MeDIP) followed by qPCR to measure DNA methylation at CpG sites around the TSSs of Crh, Avp, and Car genes. We found that METH significantly decreased cytosine methylation at CpG-rich sites near Crh and Avp TSSs but caused no significant changes near the Cart TSS. In addition, we used hydroxymethylated DNA immunoprecipitation (hMeDIP)-qPCR to test potential roles of DNA hydroxymethylation in METH-induced neuropeptide upregulation. There was significant increased DNA hydroxymethylation at the CpG-rich sequences located near Crh, Avp, and Cart TSSs. Because TET enzymes catalyze DNA hydroxymethylation, we conducted chromatin immunoprecipitation (ChIP) assay using TET1 and TET2 antibodies and found significant METH-induced increase in TET1 binding on the Crh promoter sequence. In contrast, we found no significant changes in TET1 recruitment on Avp and Cart promoter sequences. There were also no changes in TET2 binding on either Crh, Avp or Cart promoter sequences. Together, these results show a potential role of Tet1 in mediating METH-induced up-regulation of Crh mRNA expression in the nucleus accumbens. These observations hint to the possibility of using TET1 inhibitors to alleviate stress-induced neurobiological alterations in METH addicts.