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Mechanisms of Prodynorphin Gene Dysregulation in the Brain of Human Alcoholics
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Prof. Georgy Bakalkin)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The endogenous opioid system (EOS) including dynorphin opioid peptides and κ-opioid receptor (KOR) plays a critical role in alcohol dependence. Aims of the thesis were to evaluate whether the EOS undergoes molecular adaptations in brain areas involved in cognitive control of addiction in human alcohol dependent subjects, and to analyze the impact of genetic and epigenetic factors on these adaptive changes. The main findings were that (1) the dynorphin/KOR system including PDYN mRNA and dynorphins in the dorsolateral prefrontal cortex (dl-PFC), dynorphins in the hippocampus, and KOR mRNA in the orbitofrontal cortex (OFC), is upregulated in human alcoholics. No other significant changes in the EOS were found. (2) Three PDYN single nucleotide polymorphisms (SNPs), which show the most significant association with alcohol dependence, form CpG sites that are methylated in human brain at different levels. Methylation of the C, non-risk variant of the 3’-untranslated region (3’-UTR) SNP (rs2235749; C>T) was increased in dl-PFC and positively correlated with dynorphins. The DNA-binding factor that differentially targeted the T, risk allele and methylated and unmethylated C allele of this SNP was identified in human brain. We hypothesize that influences of the genetic, epigenetic and environmental factors may be integrated through alterations in methylation of the PDYN 3’jUTR CpG/SNP and, as a consequence, affect PDYN transcription and vulnerability to develop alcohol dependence. (3) 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 of the PDYN promoter SNP (rs1997794; T>C). The T, low risk allele of this SNP resides within non-canonical AP-1-binding element and may be targeted by JUND and FOSB 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. (4) It was proposed that PDYN transcription may be regulated by intragenic DNA regulatory elements controlling the DNA-protein interactions through formation of non-canonical DNA secondary structures. The dynorphin-encoding sequence in PDYN was found to have potential to form such DNA structure in vitro, and this formation was affected by CpG methylation in this region. This methylation sensitive non-canonical DNA structure formation may be involved in regulation of initiation of PDYN transcription from alternative start sites located within this region, or in splicing of non-canonical mRNA.

In conclusion, the dynorphin/KOR system has been identified as the site of robust adaptive changes associated with alcohol dependence in the areas of human brain involved in cognitive control of addiction. Regulation of PDYN was found to be brain area specific, apparently affected by the genetic and epigenetic factors, and possibly dependent on the internal properties of the gene such as its ability to form non-canonical DNA secondary structures.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. , 73 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 147
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-158235ISBN: 978-91-554-8149-0 (print)OAI: oai:DiVA.org:uu-158235DiVA: diva2:439601
Public defence
2011-10-14, BMC, B7:101, Husargatan3, Uppsala University, Sweden, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2011-09-22 Created: 2011-09-02 Last updated: 2012-05-11Bibliographically approved
List of papers
1. The endogenous opioid system in human alcoholics: molecular adaptations in brain areas involved in cognitive control of addiction
Open this publication in new window or tab >>The endogenous opioid system in human alcoholics: molecular adaptations in brain areas involved in cognitive control of addiction
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2013 (English)In: Addiction Biology, ISSN 1355-6215, E-ISSN 1369-1600, Vol. 18, no 1, 161-169 p.Article in journal (Refereed) Published
Abstract [en]

The endogenous opioid system (EOS) plays a critical role in addictive processes. Molecular dysregulations in this system may be specific for different stages of addiction cycle and neurocircuitries involved and therefore may differentially contribute to the initiation and maintenance of addiction. Here we evaluated whether the EOS is altered in brain areas involved in cognitive control of addiction including the dorsolateral prefrontal cortex (dl-PFC), orbitofrontal cortex (OFC) and hippocampus in human alcohol-dependent subjects. Levels of EOS mRNAs were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and levels of dynorphins by radioimmunoassay (RIA) in post-mortem specimens obtained from 14 alcoholics and 14 controls. Prodynorphin mRNA and dynorphins in dl-PFC, κ-opioid receptor mRNA in OFC and dynorphins in hippocampus were up-regulated in alcoholics. No significant changes in expression of proenkephalin, and µ- and δ-opioid receptors were evident; pro-opiomelanocortin mRNA levels were below the detection limit. Activation of the κ-opioid receptor by up-regulated dynorphins in alcoholics may underlie in part neurocognitive dysfunctions relevant for addiction and disrupted inhibitory control.

Keyword
Alcohol dependence, dynorphin, endogenous opioid system, human brain, κ-opioid receptor, prodynorphin
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-166255 (URN)10.1111/j.1369-1600.2011.00366.x (DOI)000312740500016 ()
Available from: 2012-01-11 Created: 2012-01-11 Last updated: 2017-12-08Bibliographically approved
2. 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
3. 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
Show others...
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
4. Conformational polymorphism of oligonucleotides with the opioid peptide dynorphin encoding sequences: effect of CpG methylation and human pathogenic mutations
Open this publication in new window or tab >>Conformational polymorphism of oligonucleotides with the opioid peptide dynorphin encoding sequences: effect of CpG methylation and human pathogenic mutations
(English)Manuscript (preprint) (Other academic)
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-158233 (URN)
Available from: 2011-09-02 Created: 2011-09-02 Last updated: 2011-11-03Bibliographically approved

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