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

Abstract

BackgroundAlcohol 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.MethodsWe 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.ResultsEleven 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.ConclusionsOur 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.

Emerging roles for ncRNAs in alcohol use disorders

“…The expression level of lncRNAs is highest in brain (Derrien et al, 2012), and similar to protein-coding genes; they are regulated by neuronal activity (Barry et al, 2014) and show cell type- and brain region-specific expression patterns (Belgard et al, 2011; Mercer, Dinger, Sunkin, Mehler, & Mattick, 2008), suggesting that their expression is important in discrete CNS functions. Although the physiological roles of lncRNAs are still emerging, they appear to be involved in cis-regulation of neighboring genes (Goff et al, 2015; Zuo et al, 2016) and the regulation of gene expression involved in adaptive behavior (Bekdash & Harrison, 2015; Spadaro et al, 2015). The extent to which specific lncRNAs contribute to the development and maintenance of alcohol- or other substance-abuse disorders is currently an under-investigated area, with the potential to uncover novel regulatory gene networks involved in the addictive process.…”

Could ethanol-induced alterations in the expression of glutamate transporters in testes contribute to the effect of paternal drinking on the risk of abnormalities in the offspring?

“…Both glutamatergic and GABAergic synapses are thought to be strongly involved in mediating the effects of alcohol [7], particularly via the NMDA-type of glutamate receptor and the alpha4-subunit containing GABA(A) receptor [8,9,10]. Both GABAergic and glutamatergic signalling systems, including the two types of receptors mentioned above, are altered in alcoholism [11,12,13,14]. Interestingly, it is not only the synaptic receptors which are affected by alcohol but also the neurotransmitter-inactivating mechanisms that are changed in alcoholic brains, particularly in the case of glutamatergic neurotransmission.…”

“…[19] and receptors [20,21]) sequestered behind such barrier would seem to imply that a signalling apparatus, possibly involving germ cells and sperm, both in their mature and immature forms either dormant or active and mediated by L-glutamate, exists and functions in testes. Glutamate transporters would then provide a regulating ("inactivating") mechanism analogous to that functioning at brain synapses (reviews: [12,13). Alternatively, Lglutamate transport, mainly by GLAST (EAAT1) and EAAT5 could trigger chloride influx thus hyperpolarizing and activating the sperm [21].…”

Sex Differences in the Expression of the α5 Subunit of the GABAA Receptor in Alcoholics with and without Cirrhosis of the Liver

“…These differences are likely to yield GABA A receptors with distinct pharmacological and functional characteristics (reviewed in Dick et al, ). The mechanism by which this occurs is not known, but previous studies have shown that the expression of selected GABA A receptor subunit can be selectively altered by miRNAs (Bekdash and Harrison, ; Zhao et al, ).…”

“…Some of these miRNAs selectively target isoforms of GABA A receptors to manipulate their expression. Specifically, miR‐181, miR‐216, and miR‐203 directly target the α1 subunit (Zhao et al, ), and miR‐155, miR‐186, miR‐24, miR‐27b, and miR‐375 directly target the α4 subunit (Bekdash and Harrison, ). Of these, only 1 miRNA, miR‐203, is up‐regulated in the prefrontal cortex of human alcoholics (Lewohl et al, ).…”