Methamphetamine Induces TET1- and TET3-Dependent DNA Hydroxymethylation of Crh and Avp Genes in the Rat Nucleus Accumbens

Methamphetamine (METH) addiction is a biopsychosocial disorder that is accompanied by multiple relapses even after prolonged abstinence, suggesting the possibilities of long-lasting maladaptive epigenetic changes in the brain. Here, we show that METH administration produced time-dependent increases in the expression of corticotropin-releasing hormone (Crh/Crf), arginine vasopressin (Avp), and cocaine- and amphetamine-regulated transcript prepropeptide (Cartpt) mRNAs in the rat nucleus accumbens (NAc). Chromatin immunoprecipitation (ChIP) assays revealed that METH increased the abundance of phosphorylated CREB (pCREB) at the promoter of Cartpt but not at Avp or Crh DNA sequences. In contrast, METH produced DNA hypomethylation at sites near the Crh transcription start site (TSS) and at intragenic Avp sequences. METH also increased DNA hydroxymethylation at the Crh TSS and at intragenic Avp sites. In addition, METH increased the protein expression of ten-eleven-translocation enzymes that catalyze DNA hydroxymethylation. Importantly, METH increased TET1 binding at the Crh promoter and increased TET3 binding at Avp intragenic regions. We further tested the role of TET enzymes in METH-induced changes in gene expression by using the TET inhibitor, 1,5-isoquinolinediol (IQD), and found that IQD blocked METH-induced increases in Crh and Avp mRNA expression. Together, these results indicate that METH produced changes in neuropeptide transcription by both activation of the cAMP/CREB pathway and stimulation of TET-dependent DNA hydroxymethylation. These results provide molecular evidence for epigenetic controls of METH-induced changes in the expression of neuropeptides.Fil: Jayanthi, Subramaniam. National Institutes of Health; Estados UnidosFil: Gonzalez, Betina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: McCoy, Michael T.. National Institutes of Health; Estados UnidosFil: Ladenheim, Bruce. National Institutes of Health; Estados UnidosFil: Bisagno, Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Cadet, Jean Lud. National Institutes of Health; Estados Unido

Compulsive methamphetamine taking in the presence of punishment is associated with increased oxytocin expression in the nucleus accumbens of rats

Methamphetamine addiction is mimicked in rats that self-administer the drug. However, these self-administration (SA) models do not include adverse consequences that are necessary to reach a diagnosis of addiction in humans. Herein, we measured genome-wide transcriptional consequences of methamphetamine SA and footshocks in the rat brain. We trained rats to self-administer methamphetamine for 20 days. Thereafter, lever-presses for methamphetamine were punished by mild footshocks for 5 days. Response-contingent punishment significantly reduced methamphetamine taking in some rats (shock-sensitive, SS) but not in others (shock-resistant, SR). Rats also underwent extinction test at one day and 30 days after the last shock session. Rats were euthanized one day after the second extinction test and the nucleus accumbens (NAc) and dorsal striatum were collected to measure gene expression with microarray analysis. In the NAc, there were changes in the expression of 13 genes in the SRvsControl and 9 genes in the SRvsSS comparison. In the striatum, there were 9 (6 up, 3 down) affected genes in the SRvsSS comparison. Among the upregulated genes was oxytocin in the NAc and CARTpt in the striatum of SR rats. These observations support a regional role of neuropeptides in the brain after a long withdrawal interval when animals show incubation of methamphetamine craving

Methamphetamine Induces Dopamine D1 Receptor-Dependent Endoplasmic Reticulum Stress-Related Molecular Events in the Rat Striatum

Methamphetamine (METH) is an illicit toxic psychostimulant which is widely abused. Its toxic effects depend on the release of excessive levels of dopamine (DA) that activates striatal DA receptors. Inhibition of DA-mediated neurotransmission by the DA D1 receptor antagonist, SCH23390, protects against METH-induced neuronal apoptosis. The initial purpose of the present study was to investigate, using microarray analyses, the influence of SCH23390 on transcriptional responses in the rat striatum caused by a single METH injection at 2 and 4 hours after drug administration. We identified 545 out of a total of 22,227 genes as METH-responsive. These include genes which are involved in apoptotic pathways, endoplasmic reticulum (ER) stress, and in transcription regulation, among others. Of these, a total of 172 genes showed SCH23390-induced inhibition of METH-mediated changes. Among these SCH23390-responsive genes were several genes that are regulated during ER stress, namely ATF3, HSP27, Hmox1, HSP40, and CHOP/Gadd153. The secondary goal of the study was to investigate the role of DA D1 receptor stimulation on the expression of genes that participate in ER stress-mediated molecular events. We thus used quantitative PCR to confirm changes in the METH-responsive ER genes identified by the microarray analyses. We also measured the expression of these genes and of ATF4, ATF6, BiP/GRP78, and of GADD34 over a more extended time course. SCH23390 attenuated or blocked METH-induced increases in the expression of the majority of these genes. Western blot analysis revealed METH-induced increases in the expression of the antioxidant protein, Hmox1, which lasted for about 24 hours after the METH injection. Additionally, METH caused DA D1 receptor-dependent transit of the Hmox1 regulator protein, Nrf2, from cytosolic into nuclear fractions where the protein exerts its regulatory functions. When taken together, these findings indicate that SCH23390 can provide protection against neuronal apoptosis by inhibiting METH-mediated DA D1 receptor-mediated ER stress in the rat striatum. Our data also suggest that METH-induced toxicity might be a useful model to dissect molecular mechanisms involved in ER stress-dependent events in the rodent brain

l-Dopa induced dyskinesias in Parkinsonian mice: Disease severity or l-Dopa history

AbstractIn Parkinson’s disease, the efficacy of l-Dopa treatment changes over time, as dyskinesias emerge with previously beneficial doses. Using MitoPark mice, that models mitochondrial failure in dopamine (DA) neurons and mimics the progressive loss of dopamine observed in Parkinson’s disease, we found that the severity of DA denervation and associated adaptations in striatal neurotransmission at the time of initiation of l-Dopa treatment determines development of l-Dopa induced dyskinesias. We treated 20-week, and 28-week old MitoPark mice with l-Dopa (10mg/kg i.p. twice a day) and found locomotor responses to be significantly different. While all MitoPark mice developed sensitization to l-Dopa treatment over time, 28-week old MitoPark mice with extensive striatal DA denervation developed abnormal involuntary movements rapidly and severely after starting l-Dopa treatment, as compared to a more gradual escalation of movements in 20-week old animals that started treatment at earlier stages of degeneration. Our data support that it is the extent of loss of DA innervation that determines how soon motor complications develop with l-Dopa treatment. Gene array studies of striatal neurotransmitter receptors revealed changes in mRNA expression levels for DA, serotonin, glutamate and GABA receptors in striatum of 28-week old MitoPark mice. Our results support that delaying l-Dopa treatment until Parkinson’s disease symptoms become more severe does not delay the development of l-Dopa-induced dyskinesias. MitoPark mice model genetic alterations known to impair mitochondrial function in a subgroup of Parkinson patients and provide a platform in which to study treatments to minimize the development of dyskinesia

Methamphetamine Self-Administration Is Associated with Persistent Biochemical Alterations in Striatal and Cortical Dopaminergic Terminals in the Rat

Methamphetamine (meth) is an illicit psychostimulant that is abused throughout the world. Repeated passive injections of the drug given in a single day or over a few days cause significant and long-term depletion of dopamine and serotonin in the mammalian brain. Because meth self-administration may better mimic some aspects of human drug-taking behaviors, we examined to what extent this pattern of drug treatment might also result in damage to monoaminergic systems in the brain. Rats were allowed to intravenously self-administer meth (yoked control rats received vehicle) 15 hours per day for 8 days before being euthanized at either 24 hours or at 7 and 14 days after cessation of drug taking. Meth self-administration by the rats was associated with a progressive escalation of daily drug intake to 14 mg/kg per day. Animals that self-administered meth exhibited dose-dependent decreases in striatal dopamine levels during the period of observation. In addition, there were significant reductions in the levels of striatal dopamine transporter and tyrosine hydroxylase proteins. There were also significant decreases in the levels of dopamine, dopamine transporter, and tyrosine hydroxylase in the cortex. In contrast, meth self-administration caused only transient decreases in norepinephrine and serotonin levels in the two brain regions, with these values returning to normal at seven days after cessation of drug taking. Importantly, meth self-administration was associated with significant dose-dependent increases in glial fibrillary acidic protein in both striatum and cortex, with these changes being of greater magnitude in the striatum. These results suggest that meth self-administration by rats is associated with long-term biochemical changes that are reminiscent of those observed in post-mortem brain tissues of chronic meth abusers

METHAMPHETAMINE PRECONDITIONING CAUSES DIFFERENTIAL CHANGES IN STRIATAL TRANSCRIPTIONAL RESPONSES TO LARGE DOSES OF THE DRUG

Methamphetamine (METH) is a toxic drug of abuse, which can cause significant decreases in the levels of monoamines in various brain regions. However, animals treated with progressively increasing doses of METH over several weeks are protected against the toxic effects of the drug. In the present study, we tested the possibility that this pattern of METH injections might be associated with transcriptional changes in the rat striatum, an area of the brain which is known to be very sensitive to METH toxicity and which is protected by METH preconditioning. We found that the presence and absence of preconditioning followed by injection of large doses of METH caused differential expression in different sets of striatal genes. Quantitative PCR confirmed METH-induced changes in some genes of interest. These include small heat shock 27 kD proteins 1 and 2 (HspB1 and HspB2), brain derived neurotrophic factor (BDNF), and heme oxygenase-1 (Hmox-1). Our observations are consistent with previous studies which have reported that ischemic or pharmacological preconditioning can cause reprogramming of gene expression after lethal ischemic insults. These studies add to the growing literature on the effects of preconditioning on the brain transcriptome

Chronic Methamphetamine Administration Causes Differential Regulation of Transcription Factors in the Rat Midbrain

Methamphetamine (METH) is an addictive and neurotoxic psychostimulant widely abused in the USA and throughout the world. When administered in large doses, METH can cause depletion of striatal dopamine terminals, with preservation of midbrain dopaminergic neurons. Because alterations in the expression of transcription factors that regulate the development of dopaminergic neurons might be involved in protecting these neurons after toxic insults, we tested the possibility that their expression might be affected by toxic doses of METH in the adult brain. Male Sprague-Dawley rats pretreated with saline or increasing doses of METH were challenged with toxic doses of the drug and euthanized two weeks later. Animals that received toxic METH challenges showed decreases in dopamine levels and reductions in tyrosine hydroxylase protein concentration in the striatum. METH pretreatment protected against loss of striatal dopamine and tyrosine hydroxylase. In contrast, METH challenges caused decreases in dopamine transporters in both saline- and METH-pretreated animals. Interestingly, METH challenges elicited increases in dopamine transporter mRNA levels in the midbrain in the presence but not in the absence of METH pretreatment. Moreover, toxic METH doses caused decreases in the expression of the dopamine developmental factors, Shh, Lmx1b, and Nurr1, but not in the levels of Otx2 and Pitx3, in saline-pretreated rats. METH pretreatment followed by METH challenges also decreased Nurr1 but increased Otx2 and Pitx3 expression in the midbrain. These findings suggest that, in adult animals, toxic doses of METH can differentially influence the expression of transcription factors involved in the developmental regulation of dopamine neurons. The combined increases in Otx2 and Pitx3 expression after METH preconditioning might represent, in part, some of the mechanisms that served to protect against METH-induced striatal dopamine depletion observed after METH preconditioning

Methamphetamine Causes Differential Alterations in Gene Expression and Patterns of Histone Acetylation/Hypoacetylation in the Rat Nucleus Accumbens

Methamphetamine (METH) addiction is associated with several neuropsychiatric symptoms. Little is known about the effects of METH on gene expression and epigenetic modifications in the rat nucleus accumbens (NAC). Our study investigated the effects of a non-toxic METH injection (20 mg/kg) on gene expression, histone acetylation, and the expression of the histone acetyltransferase (HAT), ATF2, and of the histone deacetylases (HDACs), HDAC1 and HDAC2, in that structure. Microarray analyses done at 1, 8, 16 and 24 hrs after the METH injection identified METH-induced changes in the expression of genes previously implicated in the acute and longterm effects of psychostimulants, including immediate early genes and corticotropin-releasing factor (Crf). In contrast, the METH injection caused time-dependent decreases in the expression of other genes including Npas4 and cholecystokinin (Cck). Pathway analyses showed that genes with altered expression participated in behavioral performance, cell-to-cell signaling, and regulation of gene expression. PCR analyses confirmed the changes in the expression of c-fos, fosB, Crf, Cck, and Npas4 transcripts. To determine if the METH injection caused post-translational changes in histone markers, we used western blot analyses and identified METH-mediated decreases in histone H3 acetylated at lysine 9 (H3K9ac) and lysine 18 (H3K18ac) in nuclear sub-fractions. In contrast, the METH injection caused time-dependent increases in acetylated H4K5 and H4K8. The changes in histone acetylation were accompanied by decreased expression of HDAC1 but increased expression of HDAC2 protein levels. The histone acetyltransferase, ATF2, showed significant METH-induced increased in protein expression. These results suggest that METH-induced alterations in global gene expression seen in rat NAC might be related, in part, to METH-induced changes in histone acetylation secondary to changes in HAT and HDAC expression. The causal role that HATs and HDACs might play in METH-induced gene expression needs to be investigated further

Methamphetamine Preconditioning Alters Midbrain Transcriptional Responses to Methamphetamine-Induced Injury in the Rat Striatum

Methamphetamine (METH) is an illicit drug which is neurotoxic to the mammalian brain. Numerous studies have revealed significant decreases in dopamine and serotonin levels in the brains of animals exposed to moderate-to-large METH doses given within short intervals of time. In contrast, repeated injections of small nontoxic doses of the drug followed by a challenge with toxic METH doses afford significant protection against monoamine depletion. The present study was undertaken to test the possibility that repeated injections of the drug might be accompanied by transcriptional changes involved in rendering the nigrostriatal dopaminergic system refractory to METH toxicity. Our results confirm that METH preconditioning can provide significant protection against METH-induced striatal dopamine depletion. In addition, the presence and absence of METH preconditioning were associated with substantial differences in the identity of the genes whose expression was affected by a toxic METH challenge. Quantitative PCR confirmed METH-induced changes in genes of interest and identified additional genes that were differentially impacted by the toxic METH challenge in the presence of METH preconditioning. These genes include small heat shock 27 kD 27 protein 2 (HspB2), thyrotropin-releasing hormone (TRH), brain derived neurotrophic factor (BDNF), c-fos, and some encoding antioxidant proteins including CuZn superoxide dismutase (CuZnSOD), glutathione peroxidase (GPx)-1, and heme oxygenase-1 (Hmox-1). These observations are consistent, in part, with the transcriptional alterations reported in models of lethal ischemic injuries which are preceded by ischemic or pharmacological preconditioning. Our findings suggest that multiple molecular pathways might work in tandem to protect the nigrostriatal dopaminergic pathway against the deleterious effects of the toxic psychostimulant. Further analysis of the molecular and cellular pathways regulated by these genes should help to provide some insight into the neuroadaptive potentials of the brain when repeatedly exposed to drugs of abuse

Methamphetamine-Induced Dopamine-Independent Alterations in Striatal Gene Expression in the 6-Hydroxydopamine Hemiparkinsonian Rats

Unilateral injections of 6-hydroxydopamine into the medial forebrain bundle are used extensively as a model of Parkinson's disease. The present experiments sought to identify genes that were affected in the dopamine (DA)–denervated striatum after 6-hydroxydopamine-induced destruction of the nigrostriatal dopaminergic pathway in the rat. We also examined whether a single injection of methamphetamine (METH) (2.5 mg/kg) known to cause changes in gene expression in the normally DA-innervated striatum could still influence striatal gene expression in the absence of DA. Unilateral injections of 6-hydroxydopamine into the medial forebrain bundle resulted in METH-induced rotational behaviors ipsilateral to the lesioned side and total striatal DA depletion on the lesioned side. This injection also caused decrease in striatal serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) levels. DA depletion was associated with increases in 5-HIAA/5-HT ratios that were potentiated by the METH injection. Microarray analyses revealed changes (± 1.7-fold, p<0.025) in the expression of 67 genes on the lesioned side in comparison to the intact side of the saline-treated hemiparkinsonian animals. These include follistatin, neuromedin U, and tachykinin 2 which were up-regulated. METH administration caused increases in the expression of c-fos, Egr1, and Nor-1 on the intact side. On the DA-depleted side, METH administration also increased the expression of 61 genes including Pdgf-d and Cox-2. There were METH-induced changes in 16 genes that were common in the DA-innervated and DA-depleted sides. These include c-fos and Nor-1 which show greater changes on the normal DA side. Thus, the present study documents, for the first time, that METH mediated DA-independent changes in the levels of transcripts of several genes in the DA-denervated striatum. Our results also implicate 5-HT as a potential player in these METH-induced alterations in gene expression because the METH injection also caused significant increases in 5-HIAA/5-HT ratios on the DA-depleted side