Opposing actions of ethanol and nicotine on microRNAs are mediated by nicotinic acetylcholine receptors in fetal cerebral cortical-derived neural progenitor cells

Abstract

Background: Ethanol (EtOH) and nicotine are often co-abused. However, their combined effects on fetal neural development, particularly on fetal neural stem cells (NSCs), which generate most neurons of the adult brain during the second trimester of pregnancy, are poorly understood. We previously showed that EtOH influenced NSC maturation in part, by suppressing the expression of specific microRNAs (miRNAs). Here, we tested in fetal NSCs the extent to which EtOH and nicotine coregulated known EtOH-sensitive (miR-9, miR-21, miR-153, and miR-335), a nicotine-sensitive miRNA (miR-140-3p), and mRNAs for nicotinic acetylcholine receptor (nAChR) subunits. Additionally, we tested the extent to which these effects were nAChR dependent.

Methods: Gestational day 12.5 mouse fetal murine cerebral cortical-derived neurosphere cultures were exposed to EtOH, nicotine, and mecamylamine, a noncompetitive nAChR antagonist, individually or in combination, for short (24 hour) and long (5 day) periods, to mimic exposure during the in vivo period of neurogenesis. Levels of miRNAs, miRNA-regulated transcripts, and nAChR subunit mRNAs were assessed by quantitative reverse transcription polymerase chain reaction.

Results: EtOH suppressed the expression of known EtOH-sensitive miRNAs and miR-140-3p, while nicotine at concentrations attained by cigarette smokers induced a dose-related increase in these miRNAs. Nicotine's effect was blocked by EtOH and by mecamylamine. Finally, EtOH decreased the expression of nAChR subunit mRNAs and, like mecamylamine, prevented the nicotine-associated increase in α4 and β2 nAChR transcripts.

Conclusions: EtOH and nicotine exert mutually antagonistic, nAChR-mediated effects on teratogen-sensitive miRNAs in fetal NSCs. These data suggest that concurrent exposure to EtOH and nicotine disrupts miRNA regulatory networks that are important for NSC maturation.

Figure 1

Dose-dependent effects of nicotine on the expression of (a) ethanol-sensitive miRNAs, miR-9, miR-21, miR-153 and miR-335 and (b) a previously identified nicotine-sensitive miRNA, miR-140-3p in GD12.5-derived mouse fetal cortical neuroepithelial precursors. Lower doses of nicotine, within the range attained by cigarette smokers, result in an increase in miRNA expression, whereas higher pharmacological concentrations of nicotine result in a suppression of miRNAs towards control levels. Data presented as mean (±SEM) fold-change relative to the mean of the control group (2^-ΔΔCT). Asterisks indicate statistically significant changes from controls (for details, see results section).

Figure 2

Ethanol inhibits the expression of previously identified ethanol-sensitive and nicotine-sensitive miRNAs. Data presented as mean (±SEM) fold-change relative to the mean of the control group (2^-ΔΔCT). Asterisks indicate statistically significant changes from controls (for details, see results section).

Figure 3

Regulation of miRNAs following long-term (5 day) and short-term (24 hour) ethanol/nicotine exposure. (a) Following five days exposure, ethanol and mecamylamine (MECA), individually and in combination, prevented the 1.0 uM nicotine-induced increase in miRNA expression. These data indicate that ethanol is functionally similar to MECA, a non-competitive nAChR antagonist, in terms of nAChR-mediated miRNA regulation. (b–d) Following short-term (24-hour) exposure, there was a main effect of ethanol on the expression of a sub-population of miRNAs. Ethanol alone, or in combination with nicotine, decreased the expression of miR-9 (b), miR-335 (c), and miR-140-3p (d). Data presented as mean (±SEM) fold-change relative to the mean of the control group (2^-ΔΔCT). Asterisks indicate statistically significant changes from controls (for details, see results section).

Figure 4

(a) GD12.5 mouse neuroepithelial cells express α4, α7 and β2 nAChR subunit mRNA transcripts at higher levels compared to α3 and β4 transcripts. Data is expressed as mean (±SEM) of 2^-ΔCT relative to 18s RNA (b) Nicotine induced a statistically significant and dose-related increase in the expression of mRNA transcripts for α4 and β2 nAChR subunits. Data presented as mean (±SEM) fold-change relative to the mean of the control group (2^-ΔΔCT). Asterisks indicate statistically significant changes (for details, see results section).

Figure 5

(a) Following long-term exposure, Ethanol acted as an inverse agonist in suppressing the expression of α3, α4, α7 and β4 nAChR subunit mRNAs relative to controls. Both ethanol and MECA prevented the 1.0uM nicotine-induced increase in α4 and β2 nAChR mRNAs However, concurrent administration of ethanol and MECA resulted in the reappearance of the inductive effect of nicotine, specifically on β2 mRNA transcript levels, suggesting that ethanol’s effects on nAChR mRNA transcripts is contextual, and perhaps dependent on the activation state of nAChRs. (b–e) Short-term (24-hour) exposure to nicotine produced a significant increase in the mRNA expression for α3 (b), α4 (c), β2 (d), and β4 (e) nAChRs. Ethanol prevented the nicotine-mediated increase in the mRNAs for α4 and β2 nAChRs. Data presented as mean (±SEM) fold-change relative to the mean of the control group (2^-ΔΔCT). Asterisks indicate statistically significant changes relative to controls, ‘+’ indicates significant main effect of nicotine, and ‘#’ indicates significant effect of concurrent ethanol exposure, relative to nicotine alone (for details, see results section).