Limits...
Downregulation of Gabra4 expression during alcohol withdrawal is mediated by specific microRNAs in cultured mouse cortical neurons.

Bekdash RA, Harrison NL - Brain Behav (2015)

Bottom Line: This study addressed the effects of AW on changes in the expression of Gabra4 and related genes that encode other subunits of GABAARs, and the potential regulation of Gabra4 by microRNAs.Promoter-reporter experiments supported the idea that miR-155, miR-186, miR-24, miR-27b, or miR-375 bind to the 3'UTR of Gabra4 and thereby inhibit protein production.Our data suggest that AW decreases Gabra4 expression, and that this may be mediated in part by the induction of specific microRNAs in cortical neurons during AW.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Columbia University New York, New York, 10032.

ABSTRACT

Background: Alcohol abuse and dependence are a serious public health problem. A large number of alcohol-regulated genes, (ARGs) are known to be influenced by alcohol use and withdrawal (AW), and recent evidence suggests that neuroadaptation to alcohol may be due in part to epigenetic changes in the expression of ARGs. Gabra4, which encodes the α4 subunit of GABAA receptors (GABAARs), is one of a number of ARGs that show remarkable plasticity in response to alcohol, being rapidly upregulated by acute alcohol exposure. This study addressed the effects of AW on changes in the expression of Gabra4 and related genes that encode other subunits of GABAARs, and the potential regulation of Gabra4 by microRNAs.

Methods: We studied gene and microRNAs expression, using RT-PCR and microRNA microarray in cultured cortical neurons treated with alcohol, which was then removed in order to simulate AW in vitro. We also used microRNA mimics or inhibitors, and a promoter-reporter construct carrying the 3'UTR of Gabra4.

Results: Eleven hours after removal of alcohol, Gabra4 was downregulated, with a modest increase in the expression of Gabrg2, but no change in the expression of Gabra1, Gabrd, or Gabrb2. microRNA profiling in neurons undergoing AW revealed upregulation in the expression of miR-155, miR-186, miR-24, and miR-375 after 8 h of AW. Transfection with molecular mimics of miR-186, miR-24, or miR-375 also downregulated Gabra4 expression, whereas transfection with the corresponding inhibitors of these microRNAs normalized Gabra4 expression in AW neurons to the level measured in control neurons. Promoter-reporter experiments supported the idea that miR-155, miR-186, miR-24, miR-27b, or miR-375 bind to the 3'UTR of Gabra4 and thereby inhibit protein production.

Conclusions: Our data suggest that AW decreases Gabra4 expression, and that this may be mediated in part by the induction of specific microRNAs in cortical neurons during AW.

No MeSH data available.


Related in: MedlinePlus

Conceptual diagram of the effects of miRNAs on Gabra4 expression during AW. The Mus musculus Gabra4 gene (ENSMUSG00000029211) is located on chromosome 5. The transcript (ENSMUST00000031121) is 4123 bps, has nine coding exons, 5′UTR (583 bps) and 3′UTR (1965 bps). It is translated into an alpha4 subunit (translation length = 552 residues (www.ensemble.org). 1. Alcohol withdrawal (AW) may have caused epigenetic changes in miRNA genes such as changes in DNA methylation or histone marks. 2. These alcohol-induced epigenetic changes may have altered miRNAs expression such as miR-186/miR-24. 3. Altered miRNAs could influence Gabra4 expression by binding to its 3′UTR causing mRNA degradation or 4. Translational repression or both. CH3 = methyl group; Ac = acetylation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4559018&req=5

fig10: Conceptual diagram of the effects of miRNAs on Gabra4 expression during AW. The Mus musculus Gabra4 gene (ENSMUSG00000029211) is located on chromosome 5. The transcript (ENSMUST00000031121) is 4123 bps, has nine coding exons, 5′UTR (583 bps) and 3′UTR (1965 bps). It is translated into an alpha4 subunit (translation length = 552 residues (www.ensemble.org). 1. Alcohol withdrawal (AW) may have caused epigenetic changes in miRNA genes such as changes in DNA methylation or histone marks. 2. These alcohol-induced epigenetic changes may have altered miRNAs expression such as miR-186/miR-24. 3. Altered miRNAs could influence Gabra4 expression by binding to its 3′UTR causing mRNA degradation or 4. Translational repression or both. CH3 = methyl group; Ac = acetylation.

Mentions: We experimentally confirmed that several miRNAs could regulate expression of a luciferase reporter construct that carries the 3′UTR of Gabra4. This suggests that the cumulative effects of several miRNAs are more effective on overall regulation of Gabra4 expression than the effect of a single miRNA. Luciferase assay suggested that miR-155, miR-186, miR-24, miR-27b, and miR-375 can all bind to the 3′UTR of Gabra4. The decrease in luciferase activity correlated well with the decrease in α4 subunit protein levels. This may suggest that the upregulation in expression of miR-186, miR-24 and/or miR-375 during AW modulates Gabra4 expression at the posttranscriptional level. Here, we suggest a potential molecular mechanism by which AW alters miRNAs expression and how these miRNAs may play a role in mediating the effects of AW on Gabra4 expression (Fig.10). The complexity of the regulatory system mediated by miRNAs may be better understood in the context of other regulatory mechanisms, such as DNA methylation and histone marks that may also affect Gabra4 expression. It will be interesting in future studies to explore the interactions among these epigenetic mechanisms in modulating Gabra4 expression during AW.


Downregulation of Gabra4 expression during alcohol withdrawal is mediated by specific microRNAs in cultured mouse cortical neurons.

Bekdash RA, Harrison NL - Brain Behav (2015)

Conceptual diagram of the effects of miRNAs on Gabra4 expression during AW. The Mus musculus Gabra4 gene (ENSMUSG00000029211) is located on chromosome 5. The transcript (ENSMUST00000031121) is 4123 bps, has nine coding exons, 5′UTR (583 bps) and 3′UTR (1965 bps). It is translated into an alpha4 subunit (translation length = 552 residues (www.ensemble.org). 1. Alcohol withdrawal (AW) may have caused epigenetic changes in miRNA genes such as changes in DNA methylation or histone marks. 2. These alcohol-induced epigenetic changes may have altered miRNAs expression such as miR-186/miR-24. 3. Altered miRNAs could influence Gabra4 expression by binding to its 3′UTR causing mRNA degradation or 4. Translational repression or both. CH3 = methyl group; Ac = acetylation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4559018&req=5

fig10: Conceptual diagram of the effects of miRNAs on Gabra4 expression during AW. The Mus musculus Gabra4 gene (ENSMUSG00000029211) is located on chromosome 5. The transcript (ENSMUST00000031121) is 4123 bps, has nine coding exons, 5′UTR (583 bps) and 3′UTR (1965 bps). It is translated into an alpha4 subunit (translation length = 552 residues (www.ensemble.org). 1. Alcohol withdrawal (AW) may have caused epigenetic changes in miRNA genes such as changes in DNA methylation or histone marks. 2. These alcohol-induced epigenetic changes may have altered miRNAs expression such as miR-186/miR-24. 3. Altered miRNAs could influence Gabra4 expression by binding to its 3′UTR causing mRNA degradation or 4. Translational repression or both. CH3 = methyl group; Ac = acetylation.
Mentions: We experimentally confirmed that several miRNAs could regulate expression of a luciferase reporter construct that carries the 3′UTR of Gabra4. This suggests that the cumulative effects of several miRNAs are more effective on overall regulation of Gabra4 expression than the effect of a single miRNA. Luciferase assay suggested that miR-155, miR-186, miR-24, miR-27b, and miR-375 can all bind to the 3′UTR of Gabra4. The decrease in luciferase activity correlated well with the decrease in α4 subunit protein levels. This may suggest that the upregulation in expression of miR-186, miR-24 and/or miR-375 during AW modulates Gabra4 expression at the posttranscriptional level. Here, we suggest a potential molecular mechanism by which AW alters miRNAs expression and how these miRNAs may play a role in mediating the effects of AW on Gabra4 expression (Fig.10). The complexity of the regulatory system mediated by miRNAs may be better understood in the context of other regulatory mechanisms, such as DNA methylation and histone marks that may also affect Gabra4 expression. It will be interesting in future studies to explore the interactions among these epigenetic mechanisms in modulating Gabra4 expression during AW.

Bottom Line: This study addressed the effects of AW on changes in the expression of Gabra4 and related genes that encode other subunits of GABAARs, and the potential regulation of Gabra4 by microRNAs.Promoter-reporter experiments supported the idea that miR-155, miR-186, miR-24, miR-27b, or miR-375 bind to the 3'UTR of Gabra4 and thereby inhibit protein production.Our data suggest that AW decreases Gabra4 expression, and that this may be mediated in part by the induction of specific microRNAs in cortical neurons during AW.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Columbia University New York, New York, 10032.

ABSTRACT

Background: Alcohol abuse and dependence are a serious public health problem. A large number of alcohol-regulated genes, (ARGs) are known to be influenced by alcohol use and withdrawal (AW), and recent evidence suggests that neuroadaptation to alcohol may be due in part to epigenetic changes in the expression of ARGs. Gabra4, which encodes the α4 subunit of GABAA receptors (GABAARs), is one of a number of ARGs that show remarkable plasticity in response to alcohol, being rapidly upregulated by acute alcohol exposure. This study addressed the effects of AW on changes in the expression of Gabra4 and related genes that encode other subunits of GABAARs, and the potential regulation of Gabra4 by microRNAs.

Methods: We studied gene and microRNAs expression, using RT-PCR and microRNA microarray in cultured cortical neurons treated with alcohol, which was then removed in order to simulate AW in vitro. We also used microRNA mimics or inhibitors, and a promoter-reporter construct carrying the 3'UTR of Gabra4.

Results: Eleven hours after removal of alcohol, Gabra4 was downregulated, with a modest increase in the expression of Gabrg2, but no change in the expression of Gabra1, Gabrd, or Gabrb2. microRNA profiling in neurons undergoing AW revealed upregulation in the expression of miR-155, miR-186, miR-24, and miR-375 after 8 h of AW. Transfection with molecular mimics of miR-186, miR-24, or miR-375 also downregulated Gabra4 expression, whereas transfection with the corresponding inhibitors of these microRNAs normalized Gabra4 expression in AW neurons to the level measured in control neurons. Promoter-reporter experiments supported the idea that miR-155, miR-186, miR-24, miR-27b, or miR-375 bind to the 3'UTR of Gabra4 and thereby inhibit protein production.

Conclusions: Our data suggest that AW decreases Gabra4 expression, and that this may be mediated in part by the induction of specific microRNAs in cortical neurons during AW.

No MeSH data available.


Related in: MedlinePlus