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Preproenkephalin Gene and mRNA: Studies of Structure, Function, Cocaine Responses in an Animal Model, and Genetic Association with Human Opiate Addiction
Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Biological Research on Drug Dependence.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The endogenous opioid enkephalin neuropeptides are mediators of pain perception and have been implicated in human addictions. The preproenkephalin gene and its mRNA have also provided many examples of tissue- and species-specific variations in mRNA structure produced through a variety of transcriptional and post-transcriptional mechanisms. Resultant differences in mRNA structure, in several cases, have impact on translation of enkephalin prepropeptide. The reports and discussion presented herein describe studies of the preproenkephalin gene and mRNA structure in the guinea pig, an animal that may have specific advantages for modeling the human endogenous opioid system. A guinea pig brain cDNA library was constructed and screened for clones of preproenkephalin and preprodynorphin, which were then sequenced. These studies confirmed the predicted mRNA structure that had been previously proposed based on homology with gene sequences and other methods. Multiple transcription initiation sites for each of these prepropeptide genes were also identified. Studies were conducted in the guinea pig to evaluate the effects of the administration of cocaine in a “binge” paradigm for two and seven days on preproenkephalin mRNA levels in several brain regions. “Binge” cocaine administration for seven (but not two) days resulted in differential changes in mRNA levels in different brain regions. Decreases were observed in the nucleus accumbens and hypothalamus, and increases in the frontal cortex, amygdala and hippocampus. These findings differ from those of previous rodent studies and suggest that this species may provide a useful alternative model for the study of the effects of cocaine on preproenkephalin gene expression in the human brain. Human genetic studies were also conducted in opioid-dependent (formerly heroin-addicted) and control subjects to test the hypothesis that the preproenkephalin gene is associated with heroin addiction. In two separate studies, we obtained evidence that this gene may be associated with the development of human heroin addiction.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2004. , p. 85
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 0282-7484 ; 310
Keywords [en]
Biological research on drug dependence, Neuropeptides, Endogenous opioid system, Drug addiction, Heroin, Cocaine, Gene expression, Genetic association
Keywords [sv]
Biologisk beroendeforskning
National Category
Pharmacology and Toxicology
Identifiers
URN: urn:nbn:se:uu:diva-4013ISBN: 91-554-5889-0 (print)OAI: oai:DiVA.org:uu-4013DiVA, id: diva2:164137
Public defence
2004-04-02, B22, BMC, Husargatan 3, Uppsala, 09:00
Opponent
Supervisors
Available from: 2004-03-11 Created: 2004-03-11 Last updated: 2018-01-13Bibliographically approved
List of papers
1. Primary structure of guinea pig preprodynorphin and preproenkephalin mRNAs: multiple transcription initiation sites for preprodynorphin
Open this publication in new window or tab >>Primary structure of guinea pig preprodynorphin and preproenkephalin mRNAs: multiple transcription initiation sites for preprodynorphin
2004 (English)In: Brain Research Bulletin, ISSN 0361-9230, E-ISSN 1873-2747, Vol. 63, no 2, p. 119-126Article in journal (Refereed) Published
Abstract [en]

Preprodynorphin and preproenkephalin are protein precursors from which are derived two classes of opioid neurotransmitter peptides. Dynorphin A((1-17)) is produced by proteolytic processing of prodynorphin, and processing of proenkephalin yields the enkephalin peptides. We report here on the isolation and sequencing of multiple clones for these two mRNAs from a cDNA library. Two cDNA clones of preprodynorphin contained the full-length sequence (2.35 kb) with the primary structure predicted from the guinea pig gene sequence. In contrast, one clone encoded the full-length sequence but also an additional 192 nt at the 5' end. This sequence has high homology to the 5' flanking region of the human preprodynorphin gene, and RNase protection assays demonstrated that in addition to a primary initiation site, transcription of this mRNA is initiated at several sites 160-190 nt 5' with respect to the primary site. This difference may alter translational efficiency or mRNA stability. The sequence of preproenkephalin cDNA clones confirmed the structure predicted from the gene sequence. One clone, however, contained sequences encoded by exons 2 and 3, and initiated within the first intron (intron A) of the gene. We used RNase protection mapping to assess the abundance in the brain and pituitary of preproenkephalin transcripts that initiate within intron A. These studies confirmed that the primary transcription start site is 28 nucleotides downstream from the TATAA site, and that intron A sequences are not present in significant amounts in these tissues.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-91413 (URN)10.1016/j.brainresbull.2004.01.008 (DOI)15130700 (PubMedID)
Available from: 2004-03-11 Created: 2004-03-11 Last updated: 2017-12-14Bibliographically approved
2. “Binge” cocaine differentially alters preproenkephalin mRNA levels in guinea pig brain
Open this publication in new window or tab >>“Binge” cocaine differentially alters preproenkephalin mRNA levels in guinea pig brain
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2003 (English)In: Brain Research Bulletin, ISSN 0361-9230, E-ISSN 1873-2747, Vol. 59, no 5, p. 353-357Article in journal (Refereed) Published
Abstract [en]

Male Hartley guinea pigs were administered i.p. injections of cocaine or saline for 2 or 7 days in a "binge" paradigm. RNA was isolated from dissected brain regions and levels of preproenkephalin mRNA and total RNA were quantified by RNase protection assays. Following 2 days of "binge" cocaine administration, no significant alterations in preproenkephalin mRNA levels were detected in six brain regions. Following 7 days of cocaine administration, however, lower levels of preproenkephalin mRNA were observed in the nucleus accumbens and hypothalamus of cocaine-treated animals and higher levels in the frontal cortex and amygdala. These findings differed from previous studies in the rat, so an additional experiment was performed with animals treated at the 7 day time point. For increased statistical power, data from the two experiments were combined and examined by two-way ANOVAs; in this combined analysis, increases in preproenkephalin mRNA were observed in frontal cortex, amygdala, and hippocampus, decreases were found in the nucleus accumbens and hypothalamus, with no change in thalamus, caudate putamen, or cerebellum. These observed differences between guinea pigs and rats make this species an interesting model for neurobiological studies of cocaine-induced alterations in neuropeptide gene expression in the mammalian brain.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-91414 (URN)10.1016/S0361-9230(02)00927-9 (DOI)12507685 (PubMedID)
Available from: 2004-03-11 Created: 2004-03-11 Last updated: 2017-12-14Bibliographically approved
3. A (CA)n Repeat Polymorphism in Enkephalin Gene: Association with Opiate Addiction
Open this publication in new window or tab >>A (CA)n Repeat Polymorphism in Enkephalin Gene: Association with Opiate Addiction
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Article in journal (Refereed) Submitted
Identifiers
urn:nbn:se:uu:diva-91415 (URN)
Available from: 2004-03-11 Created: 2004-03-11Bibliographically approved
4. Single nucleotide polymorphisms of the human preproenkephalin gene in association with opiate addiction
Open this publication in new window or tab >>Single nucleotide polymorphisms of the human preproenkephalin gene in association with opiate addiction
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Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-91416 (URN)
Available from: 2004-03-11 Created: 2004-03-11Bibliographically approved

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