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Epigenetic Dysregulations in the Brain of Human Alcoholics: Analysis of Opioid Genes
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Molecular neuropsychopharmacology)
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. , 84 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 209
Keyword [en]
neuropeptides, dynorphin, human brain, alcohol dependence, epigenetics, gene transcription
National Category
Natural Sciences Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-270321ISBN: 978-91-554-9445-2 (print)OAI: oai:DiVA.org:uu-270321DiVA: diva2:890827
Public defence
2016-02-26, B/B7:113a, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2016-02-04 Created: 2015-12-27 Last updated: 2016-02-12
List of papers
1. Prodynorphin CpG-SNPs associated with alcohol dependence: elevated methylation in the brain of human alcoholics
Open this publication in new window or tab >>Prodynorphin CpG-SNPs associated with alcohol dependence: elevated methylation in the brain of human alcoholics
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2011 (English)In: Addiction Biology, ISSN 1355-6215, E-ISSN 1369-1600, Vol. 16, no 3, 499-509 p.Article in journal (Refereed) Published
Abstract [en]

The genetic, epigenetic and environmental factors may influence the risk for neuropsychiatric disease through their effects on gene transcription. Mechanistically, these effects may be integrated through regulation of methylation of CpG dinucleotides overlapping with single-nucleotide polymorphisms (SNPs) associated with a disorder. We addressed this hypothesis by analyzing methylation of prodynorphin (PDYN) CpG-SNPs associated with alcohol dependence, in human alcoholics. Postmortem specimens of the dorsolateral prefrontal cortex (dl-PFC) involved in cognitive control of addictive behavior were obtained from 14 alcohol-dependent and 14 control subjects. Methylation was measured by pyrosequencing after bisulfite treatment of DNA. DNA binding proteins were analyzed by electromobility shift assay. Three PDYN CpG-SNPs associated with alcoholism were found to be differently methylated in the human brain. In the dl-PFC of alcoholics, methylation levels of the C, non-risk variant of 3'-untranslated region (3'-UTR) SNP (rs2235749; C > T) were increased, and positively correlated with dynorphins. A DNA-binding factor that differentially targeted the T, risk allele and methylated and unmethylated C allele of this SNP was identified in the brain. The findings suggest a causal link between alcoholism-associated PDYN 3'-UTR CpG-SNP methylation, activation of PDYN transcription and vulnerability of individuals with the C, non-risk allele(s) to develop alcohol dependence.

Keyword
alcohol dependence, CpG-SNPs, DNA methylation, epigenetics, prodynorphin, single-nucleotide polymorphisms
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-156264 (URN)10.1111/j.1369-1600.2011.00323.x (DOI)000292098900013 ()
Available from: 2011-07-18 Created: 2011-07-18 Last updated: 2017-12-08
2. Prodynorphin promoter SNP associated with alcohol dependence forms noncanonical AP-1 binding site that may influence gene expression in human brain
Open this publication in new window or tab >>Prodynorphin promoter SNP associated with alcohol dependence forms noncanonical AP-1 binding site that may influence gene expression in human brain
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2011 (English)In: Brain Research, ISSN 0006-8993, E-ISSN 1872-6240, Vol. 1385, 18-25 p.Article in journal (Refereed) Published
Abstract [en]

Single nucleotide polymorphism (rs1997794) in promoter of the prodynorphin gene (PDYN) associated with alcohol-dependence may impact PDYN transcription in human brain. To address this hypothesis we analyzed PDYN mRNA levels in the dorsolateral prefrontal cortex (dl-PFC) and hippocampus, both involved in cognitive control of addictive behavior and PDYN promoter SNP genotype in alcohol-dependent and control human subjects. The principal component analysis suggested that PDYN expression in the dl-PFC may be related to alcoholism, while in the hippocampus may depend on the genotype. We also demonstrated that the T, low risk SNP allele resides within noncanonical AP-1-binding element that may be targeted by JUND and FOSS proteins, the dominant AP-1 constituents in the human brain. The T to C transition abrogated AP-1 binding. The impact of genetic variations on PDYN transcription may be relevant for diverse adaptive responses of this gene to alcohol.

Keyword
Alcohol dependence, Endogenous opioid system, Prodynorphin, Gene polymorphism
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-151563 (URN)10.1016/j.brainres.2011.02.042 (DOI)000289810800003 ()21338584 (PubMedID)
Available from: 2011-04-13 Created: 2011-04-13 Last updated: 2017-12-11Bibliographically approved
3. Expression of pronociceptin and its receptor is downregulated in the brain of human alcoholics
Open this publication in new window or tab >>Expression of pronociceptin and its receptor is downregulated in the brain of human alcoholics
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2009 (English)In: Brain Research, ISSN 0006-8993, E-ISSN 1872-6240, Vol. 1305, no Suppl. 1, S80-85 p.Article in journal (Refereed) Published
Abstract [en]

Animal studies demonstrated a role of neuropeptide nociceptin (NC) and its receptor (opiate receptor like-1, OPRL1) in ethanol-induced reward; activation of the OPRL1 by natural or synthetic ligands reduced ethanol self-administration and prevented relapse to ethanol drinking. The endogenous NC may function in neuronal circuits involved in reinforcing or conditioning effects of ethanol as a "brake" to limit ethanol intake (Roberto, M., Siggins, G.R. 2006. Nociceptin/orphanin FQ presynaptically decreases GABAergic transmission and blocks the ethanol-induced increase of GABA release in central amygdala. Proc. Natl. Acad. Sci. USA 103. 9715-9720), whereas repeated ethanol intake may downregulate the endogenous NC/OPRL1 system resulting in activation of ethanol consumption. To address this hypothesis, we evaluated whether expression of the pronociceptin (PNOC) and OPRL1 genes is altered in human alcoholics. mRNAs transcribed from these genes were analyzed by quantitative RT-PCR in the prefrontal and orbitofrontal cortices, central amygdala and hippocampal dentate gyrus, structures controlling alcohol consumption. Reduction in PNOC mRNA (1.7-fold) was found in the hippocampus of alcoholics, whereas OPRL1 mRNA levels were decreased (1.4-fold) in the central amygdala. No changes in expression of these genes in other brain areas analyzed were evident. We hypothesise that chronic ethanol intake downregulates PNOC and OPRL1 gene expression in the hippocampus and amygdala, respectively. The findings may be also interpreted as inherited molecular differences between alcoholics and controls. The PNOC/OPRL1 downregulation may underlie impairment of cognitive control over alcohol seeking in alcoholics. Stimulation of the OPRL1 receptors with synthetic agonists may increase threshold for activation of ethanol-related behaviour by environmental cues, and thus may reduce cue- or stress-primed relapse to ethanol consumption.

Keyword
Alcoholism, nociceptin, OPRL1, amygdala, hippocampus
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-119998 (URN)10.1016/j.brainres.2009.05.067 (DOI)000273202100010 ()19501074 (PubMedID)
Available from: 2010-03-08 Created: 2010-03-04 Last updated: 2017-12-12Bibliographically approved
4. Neuronal Expression of Opioid Gene is Regulated by Genetically Controlled Epigenetic and Transcriptional Mechanisms in Addicted Human Brain
Open this publication in new window or tab >>Neuronal Expression of Opioid Gene is Regulated by Genetically Controlled Epigenetic and Transcriptional Mechanisms in Addicted Human Brain
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(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-270323 (URN)
Available from: 2016-01-04 Created: 2015-12-27 Last updated: 2016-02-12

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