DNA metüülimise roll kokaiini poolt põhjustatud käitumusliku sensitisatsiooni kujunemises

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Ravimsõltuvust iseloomustab vastupandamatu tung tarbida psühhotroopseid aineid hoolimata sellega kaasuvatest tõsistest kõrvaltoimetest. Uuringud on näidanud, et ravimsõltuvus kujuneb välja järk-järgult ja sõltuvusega seotud neurobioloogilised muutused võivad jääda püsima ka pärast psühhotroopsete ainete tarvitamise lõppu. Korduv psühhostimulaatori (nt. kokaiini) manustamine põhjustab katseloomadel suurenenud käitumusliku vastuse. Sellist fenomeni nimetatakse psühhomotoorseks ehk käitumuslikuks sensitisatsiooniks ning see modelleerib adiktiivset käitumist ja psühhostimulaatorite psühhootilisi komplikatsioone inimesel. Kuna käitumuslikule sensitisatsioonile on iseloomulikud püsivad muutused katseloomade käitumises, siis arvatakse, et osaliselt on selle põhjuseks lühi- ja pikaajalised geeniekspressiooni muutused, mis omakorda mõjutavad närviimpulsi ülekannet, sünapsite moodustamist ja närviringide funktsioneerimist. Mitmed uuringud viitavad, et epigeneetilised mehhanismid, eriti geeni promootori piirkonna DNA metüülimine, mille korral liidetakse DNA metüültransferaasi (DNMT) vahendusel metüülrühm tsütosiin-guanosiin (CpG) dinukleotiidsele järjestusele, on seotud pikaajaliste geeniekspressiooni muutustega. Käesoleva töö eesmärgiks oli uurida DNA metüülimise rolli kokaiini poolt põhjustatud käitumusliku sensitisatsiooni kujunemises hiirtel ja rottidel. Töö tulemused näitasid, et kokaiini manustamine suurendas dünaamiliselt Dnmt3a ja Dnmt3b ekspressiooni täiskasvanud hiirte naalduvas tuumas (nucleus accumbens) ja hipokampuses; põhjustas valitud markergeenide promootori piirkonnas nii DNA metüülimist kui ka demetüülimist ning DNMT inhibiitori, zebulariini, manustamine normaliseeris hüpermetüülitud geeni transkriptsiooni täiskasvanud hiirte naalduvas tuumas ja pidurdas käitumusliku sensitisatsiooni teket. Samuti leidsime, et keskkonnategurid, nagu metüülrühma doonor S-adenosüülmetioniin (SAM) ja varajases elueas kogetud stress, võivad DNA metüülimise kaudu soodustada psühhostimulaatoritest tingitud ravimsõltuvuse teket nii hiirtel kui ka rottidel.Drug addiction is a chronic relapsing disorder characterised by a pattern of compulsive drug seeking and taking behaviour despite severe adverse consequences. Prolonged abuse of drugs, such as psychostimulants, may contribute to behavioural abnormalities that can last for months or even years after discontinuing drug consumption. Repeated administration of psychostimulants (such as cocaine) induces an enhanced behavioural response to subsequent drug exposure, a phenomenon known as psychomotor or behavioural sensitisation. Psychostimulant-induced behavioural sensitisation in rodents provides a model for addictive behaviours, such as those associated with craving and relapse, and for the psychotic complications of psychostimulant abuse. Behavioural sensitisation is remarkably persistent phenomenon. In rodents, it can persist from months to years after drug treatment is discontinued. Persistent behavioural sensitisation indicates that drug-induced short- and long-term changes in gene expression may be involved. Accumulating data suggest that epigenetic mechanisms, such as DNA methylation (catalysed by DNA methyltransferases - DNMTs), are critical regulators of persistent gene expression changes and may be related to behavioural disorders. The aim of this study was to investigate the role of DNA methylation in the development of cocaine-induced behavioural sensitisation in mice and rats. Our data demonstrated that cocaine treatment caused a dynamic increase in Dnmt3a and Dnmt3b expression levels in the nucleus accumbens (NAc) and hippocampus of adult mice; induced both DNA methylation/demethylation in the promoter regions of the selected genes; and intracerebroventricular treatment with the DNMT inhibitor zebularine normalised hypermethylated gene transcription in the NAc of adult mice and delayed the development of cocaine-induced behavioural sensitisation. We also found that environmental factors, such as methyl group donor S-adenosylmethionine (SAM) and early life stress, may promote, via DNA methylation, the development of psychostimulant-induced drug addiction in mice and rats

Repeated Ethanol Exposure Alters DNA Methylation Status and Dynorphin/Kappa-Opioid Receptor Expression in Nucleus Accumbens of Alcohol-Preferring AA Rats

Growing evidence suggests that epigenetic mechanisms, such as DNA methylation and demethylation, and histone modifications, are involved in the development of alcohol and drug addiction. However, studies of alcohol use disorder (AUD) that are focused on epigenetic DNA modifications and gene expression changes remain conflicting. Our aim was to study the effect of repeated ethanol consumption on epigenetic regulatory enzymes such as DNA methyltransferase and demethylase enzymes and whether those changes affected dynorphin/kappa-opioid receptor system in the Nucleus Accumbens (NAc). Two groups of male alcohol-preferring Alko Alcohol (AA) rats, rats which are selectively bred for high voluntary alcohol consumption and one group of male Wistar rats were used. The first group of AA rats had access to alcohol (10% ethanol solution) for 90 min on Mondays, Wednesdays and Fridays over a period of 3 weeks to establish a stable baseline of ethanol intake (AA-ethanol). The second group of AA rats (AA-water) and the Wistar rats (Wistar-water) were provided with water. Using qPCR, we found that voluntary alcohol drinking increased Dnmt1, -3a, and -3b mRNA levels and did not affect Tet family transcripts in the AA-ethanol group when compared with AA- and Wistar-water rats. DNMT and TET enzymatic activity measurements showed similar results to qPCR, where DNMT activity was increased in AA-ethanol group compared with AA-water and Wistar-water groups, with no statistically significant difference between groups in TET enzyme activity. In line with previous data, we found an increased percentage of global DNA methylation and hydroxymethylation in the AA-ethanol group compared with control rats. Finally, we investigated changes of selected candidate genes from dynorphin/kappa-opioid receptor system (Pdyn, Kor) and Dnmt3a genes that might be important in AUD-related behaviour. Our gene expression and promoter methylation analysis revealed a significant increase in the mRNA levels of Pdyn, Kor, and Dnmt3a in the AA-ethanol group, however, these changes can only be partially associate with the aberrant DNA methylation in promoter areas of the selected candidate genes. Thus, our findings suggest that the aberrant DNA methylation is rather one of the several mechanisms involved in gene expression regulation in AA rat model.Peer reviewe

DNA Methylation Regulates Cocaine-Induced Behavioral Sensitization in Mice

The behavioral sensitization produced by repeated cocaine treatment represents the neural adaptations underlying some of the features of addiction in humans. Cocaine administrations induce neural adaptations through regulation of gene expression. Several studies suggest that epigenetic modifications, including DNA methylation, are the critical regulators of gene expression in the adult central nervous system. DNA methylation is catalyzed by DNA methyltransferases (DNMTs) and consequent promoter region hypermethylation is associated with transcriptional silencing. In this study a potential role for DNA methylation in a cocaine-induced behavioral sensitization model in mice was explored. We report that acute cocaine treatment caused an upregulation of DNMT3A and DNMT3B gene expression in the nucleus accumbens (NAc). Using methylated DNA immunoprecipitation, DNA bisulfite modification, and chromatin immunoprecipitation assays, we observed that cocaine treatment resulted in DNA hypermethylation and increased binding of methyl CpG binding protein 2 (MeCP2) at the protein phosphatase-1 catalytic subunit (PP1c) promoter. These changes are associated with transcriptional downregulation of PP1c in NAc. In contrast, acute and repeated cocaine administrations induced hypomethylation and decreased binding of MeCP2 at the fosB promoter, and these are associated with transcriptional upregulation of fosB in NAc. We also found that pharmacological inhibition of DNMT by zebularine treatment decreased cocaine-induced DNA hypermethylation at the PP1c promoter and attenuated PP1c mRNA downregulation in NAc. Finally, zebularine and cocaine co-treatment delayed the development of cocaine-induced behavioral sensitization. Together, these results suggest that dynamic changes of DNA methylation may be an important gene regulation mechanism underlying cocaine-induced behavioral sensitization