Immediate-early alcohol-responsive miRNA expression in <i>Drosophila</i>

<p>At the core of the changes characteristic of alcoholism are alterations in gene expression in the brain of the addicted individual. These changes are believed to underlie some of the neuroadaptations that promote compulsive drinking. Unfortunately, the mechanisms by which alcohol consumption produces changes in gene expression remain poorly understood. MicroRNAs (miRNAs) have emerged as important regulators of gene expression because they can coordinately modulate the translation efficiency of large sets of specific mRNAs. Here, we investigate the early miRNA responses elicited by an acute sedating dose of alcohol in the <i>Drosophila</i> model organism. In our analysis, we combine the power of next-generation sequencing with <i>Drosophila</i> genetics to identify alcohol-sensitive miRNAs and to functionally test them for a role in modulating alcohol sensitivity. We identified 14 known <i>Drosophila</i> miRNAs, and 13 putative novel miRNAs that respond to an acute sedative exposure to alcohol. Using the GeneSwitch Gal4/UAS system, a subset of these ethanol-responsive miRNAs was functionally tested to determine their individual contribution in modulating ethanol sensitivity. We identified two microRNAs that when overexpressed significantly increased ethanol sensitivity: miR-6 and miR-310. MicroRNA target prediction analysis revealed that the different alcohol-responsive miRNAs target-overlapping sets of mRNAs. Alcoholism is the product of accumulated cellular changes produced by chronic ethanol consumption. Although all of the changes described herein are extremely rapid responses evoked by a single ethanol exposure, understanding the gene expression changes that occur in the first few minutes after ethanol exposure will help us to categorize ethanol responses into those that are near instantaneous and those that are emergent responses produced only by repeated ethanol exposure.</p

The time course analysis of <i>slo</i> transcription in <i>slo</i>^∆6b flies.

<p>The mRNA levels of <i>slo</i> were determined by real-time RT-PCR using C1 primers that amplify only neural <i>slo</i> transcripts. One-way ANOVA with Dunnett’s comparison post test, n=3. Error bars represent SEM.</p

The <i>slo</i>^∆6b mutation specifically affects drug-induced neural expression of <i>slo</i>.

<p><b>A</b>) Six hours after benzyl alcohol sedation induction slo expression is increased in the wild type (CS) and <i>slo</i>^∆6b mutants; however, induction is greater in the mutant than in the wild type. <b>B</b>) The relative abundance of <i>slo</i> mRNA was not statistically different from the baseline abundance 24 h after sedation for either the wild type or the <i>slo</i>^∆6b mutant. Unpaired <i>t</i>-test, n=3. Error bars represent SEM.</p

Electrophysiological analysis of wild type and <i>slo</i>^∆6b stocks.

<p><b>A</b>) Electroconvulsive stimuli (ES) induce a stereotypical seizure response from the giant fiber pathway. Constant low-frequency stimulations were applied continuously to assess the state of responsivity of the giant fiber pathway. A seizure consists of a high-frequency initial discharge (ID) followed by a period in which the giant fiber fails to respond to stimulation (Failure) followed by a delayed discharge (DD) and then by a recovery of normal responsivity. The seizure threshold was identified by delivering electroconvulsive shock of varying voltage until the production of a stereotypical seizure response. <b>B</b>) The <i>slo</i>^∆6b mutant exhibited a significant lower average seizure threshold compared to wild-type flies. Benzyl alcohol sedation reduced stimulus voltage in both stocks. <b>C</b>) The drug-induced reduction in seizure threshold was greater in <i>slo</i>^∆6b mutants than in wild-type animals one day after sedation. <b>D</b>,<b>E</b>) The average seizure stimulus voltages of both WT and <i>slo</i>^∆6b returned to baseline at 7 d after sedation. <b>F</b>) Time course of seizure threshold after drug sedation. Re-plot of the data presented in panels B-E to illustrate the convergence of seizure threshold as a function of age. Wild-type (WT) and <i>slo</i>^∆6b lines are compared before and after benzyl alcohol (BA) sedation. Unpaired Student’s t-test, P values and number of repeats as shown. Error bars show SEM.</p

The <i>slo</i>^∆6b mutant shows unusually long-lived benzyl alcohol tolerance.

<p><b>A</b>) Schematic of the paradigm used to determine the time course of functional tolerance. A stock shows tolerance if it recovers more rapidly from its second sedation than from its first sedation. A population of age-matched females were separated into two groups. One group was mock sedated and the second group was sedated with benzyl alcohol. The animals were then housed in separate vials with food for the time intervals in panel B (1d -28 d) and then both groups were benzyl alcohol sedated in tandem, moved to fresh air (t=0), and their recovery recorded. <b>B</b>) The recovery curves describe the percentage of flies recovering after a single sedation (blue) and after a two sequential sedations (red) that were separated by the time interval shown. Tolerance lasts for at least 28 d in <i>slo</i>^∆6b; however, it is detected in wild type for only a week. Error bars represent SEM, but significance difference between curves is determined by log-rank analysis (n=4–6. *<i>P</i> ≤ 0.05).</p

State of histone H4 acetylation across the <i>slo</i> transcriptional control region after benzyl alcohol sedation.

<p><b>A</b>) Map of the <i>slo</i> transcriptional control region and areas assayed by the chromatin immunoprecipitation assay. Arrowheads identify the position of the tissue-specific <i>slo</i> core promoters, and open boxes on the line represent exons. The gray boxes below the line show the conserved elements tested in the chromatin immunoprecipitation assay. In <i>slo</i>^∆6b, the 6b site was replaced by a loxP element. <b>B</b>) H4 acetylation levels 6 h detected in WT and <i>slo</i>^∆6b after benzyl alcohol sedation. One-way ANOVA with Dunnett’s comparison post test. n=3. From left to right * signifies P=0.0458, 0.0278, and 0.0200. *** signifies P=0.0008. Fold change of acetylation was the ratio of the acetylation levels of drug-sedated flies over untreated ones. <b>C</b>) Acetylation state surveyed 24 h after BA sedation. One-way ANOVA with Dunnett’s comparison post test. n=3. From left to right * signifies P=0.0387, 0.0486, and 0.0239. Error bars represent SEM.</p

<i>slo</i>^∆6b flies are normal with respect to most behaviors.

<p>The <i>slo</i>^∆6b strain was backcrossed to the Canton S wild-type stock (WT) for six generations prior to analysis. The locomotor activity, (<b>A</b>), walking speed (<b>B</b>), climbing (<b>C</b>) and flight (<b>D</b>) of <i>slo</i>^∆6b are similar to WT, while the <i>slo</i>⁴ null mutants are deficient in all four behaviors. <b>E</b>) Circadian activity. LD entrained flies were transferred to DD and monitored. WT and <i>slo</i>^∆6b flies demonstrated rhythmic oscillation in activity. <i>slo</i>^∆6b had slightly higher peak activity than WT. However, the <i>slo</i>⁴ null mutants were arrhythmic. n=25. <b>F</b>) Percentage of WT, <i>slo</i>^∆6b, and <i>slo</i>⁴ identified as rhythmic. <b>G</b>) <i>slo</i>^∆6b mutants had a normal circadian period length. n=25. <b>H</b>) The <i>slo</i>^∆6b mutation does not alter drug resistance. Age-matched females were BA sedated and their recovery rate was determined. n=6. <b>I</b>) The <i>slo</i>^∆6b mutation, does not disturb the splicing out of the intron in which it is located. RT-PCR shows that the flanking exons are spliced normally. <b>J</b>) The <i>slo</i>^∆6b allele shows normal basal expression level (P=0.859; n=3). mRNA abundance was measured by RT-qPCR using primers specific for neuronal <i>slo</i> transcripts. <b>Statistical tests</b>. A, B, C, and F–one-way ANOVA with Dunnett’s comparison post test. *** indicates P≤ 0.001. N=25 (A), 3 (B), and 4 (C). For H, the log-rank test was used to evaluate significance between the recovery curves. For J, an unpaired <i>t</i>-test was used to evaluate significance. All error bars are SEM.</p

Modification of the <i>slo</i> transcriptional control region.

<p><b>A</b>) Transcriptional control region of <i>slo</i>. Labelled arrows are tissue-specific transcription start sites of the previously-described promoters [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075549#B11" target="_blank">11</a>]. Blue boxes represent alternative 5' exons that are unique products of each promoter. Rightmost box on the line represents the first exon common to all <i>slo</i> transcripts. Neuronal splice variants begin translation in this exon. The remainder of the coding region is not shown. Boxes below the line are non-coding conserved DNA elements [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075549#B11" target="_blank">11</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075549#B40" target="_blank">40</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075549#B41" target="_blank">41</a>]. The table summarizes benzyl alcohol induced histone H4 hyper-acetylation, neural expression of <i>slo</i>, and functional tolerance (data from Wang et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075549#B6" target="_blank">6</a>]). Plusses correspond to the H4 hyperacetylation. Arrows reflect relative abundance of <i>slo</i> mRNA (RNA). Checks identify when behavioral tolerance can be detected in the first 48 h (Tol). <b>B</b>) Homologous recombination occurred between the replacement DNA (red) and its chromosomal counterpart (blue) replaces 6b with a floxed mini-white gene. Cre recombinase was used to excise the loxP-flanked <i>white</i> gene to produce the <i>slo</i>^∆6b allele in which the 6b element has been replaced by a loxP site. <b>C</b>) Crossing scheme for 6b targeting. <b>D</b>) Products produced at the different steps in the crossing scheme described in panel C<b>. E</b>) Southern blotting confirms homologous recombination into the <i>slo</i> locus. Restriction maps of wild type, <i>slo</i>^w∆6b<i>, and </i><i>slo</i>^∆6b. Probe indicated above maps. The recipient line (WT), produces an 8.5 kb band. The <i>slo</i>^w∆6b and <i>slo</i>^∆6b recombinants produce bands whose size is indicative of high fidelity homologous recombination into at the position of the 6b element (cf. panels D and E).</p

Clustering analysis by gene-expression patterns of genes identified in this study.

<p>Genes were clustered by Pearson correlation analysis of their mRNA expression patterns induced by 21 different environmental conditions. Shades of blue in heat map denote gene-expression levels for each condition, normalized for each gene using the sum of squares of all conditions (white is lowest, dark blue highest). After clustering, genes segregate into seven distinct clusters, four of which are highly correlated (r>0.65). These clusters are denoted by red brackets. Clusters with low or no correlation (r<0.3) are denoted by gray brackets. In this study, eighteen genes (16 of which fall within the highly correlated clusters, marked in bold) were tested for their role in behavioral alcohol tolerance. Of these, ten (marked in red) significantly reduced tolerance to alcohol, while eight (only six shown) had no effect (marked in blue).</p

Genes tested for drug tolerance using mutant analysis.

<p>(#) Cluster number. (+) Significantly blocked or reduced tolerance. (−) No effect on tolerance. (L) Lethal.</p