Epigenetic Dysregulations in the Brain of Human Alcoholics Analysis of Opioid Genes

Sammanfattning: Neuropeptides are special in their expression profiles restricted to neuronal subpopulations and low tissue mRNA levels. Genetic, epigenetic and transcriptional mechanisms that define spatiotemporal expression of the neuropeptide genes have utmost importance for the formation and functions of neural circuits in normal and pathological human brain. This thesis focuses on regulation of transcription of the opioid/nociceptin genes, the largest neuropeptide family, and on identification of adaptive changes in these mechanisms associated with alcoholism as model human pathology. Two epigenetic mechanisms, the common for most cells in the dorsolateral prefrontal cortex (dlPFC) and the neuron-subpopulation specific that may orchestrate prodynorphin (PDYN) transcription in the human dlPFC have been uncovered. The first, repressive mechanism may operate through control of DNA methylation/demethylation in a short, nucleosome size promoter CpG island (CGI). The second mechanism may involve USF2, the sequence–specific methylation–sensitive transcription factor which interaction with its target element in the CpG island results in USF2 and PDYN co-expression in the same neurons. The short PDYN promoter CGI may function as a chromatin element that integrates cellular and environmental signals through changes in methylation and transcription factor binding. Alterations in USF2–dependent PDYN transcription are affected by the promoter SNP (rs1997794: T>C) under transition to pathological state, i.e. in the alcoholic brain. This and two other PDYN SNPs that are most significantly associated with alcoholism represent CpG-SNPs, which are differentially methylated in the human dlPFC. The T, low risk allele of the promoter SNP forms a noncanonical AP-1–binding element. JUND and FOSB proteins, which may form homo- or heterodimers have been identified as dominant constituents of AP-1 complex. The C, non-risk variant of the PDYN 3′ UTR SNP (rs2235749 SNP: C>T) demonstrated significantly higher methylation in alcoholics compared to controls. PDYN mRNA and dynorphin levels significantly and positively correlated with methylation of the PDYN 3′ UTR CpG-SNP suggesting its involvement in PDYN regulation. A DNA–binding factor with differential binding affinity for the T allele and methylated and unmethylated C alleles of the PDYN 3′ UTR SNP (the T allele specific binding factor, Ta-BF) has been discovered, which may function as a regulator of PDYN transcription. These findings emphasize the complexity of PDYN regulation that determines its expression in specific neuronal subpopulations and suggest previously unknown integration of epigenetic, transcriptional and genetic mechanisms that orchestrate alcohol–induced molecular adaptations in the human brain. Given the important role of PDYN in addictive behavior, the findings provide a new insight into fundamental molecular mechanisms of human brain disorder. In addition to PDYN in the dlPFC, the PNOC gene in the hippocampus and OPRL1 gene in central amygdala that were downregulated in alcoholics may contribute to impairment of cognitive control over alcohol seeking and taking behaviour.