microRNA Expression Profiles in the Ventral Hippocampus during Pubertal Development and the Impact of Peri-Pubertal Binge Alcohol Exposure

Abstract

Adolescence is hallmarked by two parallel processes of sexual maturation and adult patterning of the brain. Therefore, adolescence represents a vulnerable postnatal period for neurodevelopment where exogenous factors can negatively impact adult brain function. For example, alcohol exposure during pubertal development can lead to long-term and widespread neurobiological dysfunction and these effects have been shown to persist even in the absence of future alcohol exposure. However, the molecular mechanisms mediating the persistent effects of alcohol are unclear. We propose that dysregulation of microRNAs (miR) could be a unifying epigenetic mechanism underlying these widespread long-term changes. We tested the hypothesis that repeated alcohol exposure during pubertal development would cause disruption of normal miR expression profiles during puberty and, subsequently, their downstream mRNA target genes in the ventral hippocampus using an established rat model of adolescent binge drinking. We found 6 alcohol-sensitive miRs that were all downregulated following alcohol exposure and we also investigated the normal age-dependent changes in those miRs throughout the pubertal period. Interestingly, these miRs were normally decreased throughout the process of puberty, but alcohol prematurely exacerbated the normal decline in miR expression levels. The work presented herein provides foundational knowledge about the expression patterns of miRs during this critical period of neurodevelopment. Further, this regulation of miR and mRNA expression by alcohol exposure presents a complex regulatory mechanism by which perturbation in this time-sensitive period could lead to long-term neurological consequences.

Keywords: adolescence; alcohol; gene regulation; microRNA; neurodevelopment; puberty.

Figure 1

Animal paradigm and hormone levels across pubertal development. (A) Animal paradigm for Experiment 1 (untreated) and Experiment 2 (repeated binge ethanol exposure). Dotted lines indicate day of euthanasia. Plasma hormone levels of (B) luteinizing hormone (LH), (C) follicle-stimulating hormone (FSH), and (D) testosterone (T) in pre-, peri- and post-pubertal male rats (post-natal day (PND) 30, 44, and 74, respectively). Data are depicted as mean ± SEM, N = 10/age group.

Figure 2

Representative images of testes morphology across male pubertal development. Histological cross-sections of testes stained with hematoxylin and eosin at (A) PND 30, (B) PND 44, and (C) PND 74 (N = 3/ age group). Arrows point to primary (Ps) spermatocytes, spermatids (St) and spermatozoa (Sz). (D) Table summarizing morphological differences in testes between PND 30, PND 44, and PND 74. Morphological markers are indicated as present (+) or absent (−) in 100% of animals within an age group. Morphological markers present in less than 100% of animals within an age group indicated as (−/+).

Figure 3

Ethanol treatment during peri-puberty alters microRNA (miR) expression at PND 44. Effects of repeated binge-pattern EtOH exposure on miR levels in the ventral hippocampus of male rats at PND 44 following repeated binge-pattern EtOH administration paradigm during peri-puberty. Data analyzed by ^ΔΔCt method and depicted as mean ± SEM. N = 10/group, an (*) indicates p < 0.05.

Figure 4

Ethanol treatment during peri-puberty alters validated mRNA targets of EtOH-sensitive microRNAs (miRs) at PND 44. (A) Table of published validated mRNA targets for identified EtOH-sensitive miRs. (B) mRNA expression levels in the ventral hippocampus of male rats at PND 44 following repeated binge-pattern EtOH administration paradigm during peri-puberty. Data analyzed by ^ΔΔCt method and depicted as mean ± SEM. N = 10/group, an (*) indicates p < 0.05. (C) Western blot and (D) relative quantification of protein expression in ventral hippocampus tissue of the target mRNAs increased by EtOH. Data depicted as relative mean compared to β-Tubulin ± SEM, N = 5/group, an (*) indicates p < 0.05.

Figure 4

Ethanol treatment during peri-puberty alters validated mRNA targets of EtOH-sensitive microRNAs (miRs) at PND 44. (A) Table of published validated mRNA targets for identified EtOH-sensitive miRs. (B) mRNA expression levels in the ventral hippocampus of male rats at PND 44 following repeated binge-pattern EtOH administration paradigm during peri-puberty. Data analyzed by ^ΔΔCt method and depicted as mean ± SEM. N = 10/group, an (*) indicates p < 0.05. (C) Western blot and (D) relative quantification of protein expression in ventral hippocampus tissue of the target mRNAs increased by EtOH. Data depicted as relative mean compared to β-Tubulin ± SEM, N = 5/group, an (*) indicates p < 0.05.

Figure 5

Ethanol treatment during peri-puberty alters mRNA expression at PND 44. Effects of repeated binge-pattern EtOH exposure on mRNA levels in the ventral hippocampus of male rats at PND 44 following repeated binge-pattern EtOH administration paradigm during peri-puberty. Genes grouped by function, listed below the X-axis. Data analyzed by ^ΔΔCt method and depicted as mean ± SEM. N = 10/group, an (*) indicates p < 0.05.

Figure 6

microRNA (miR) expression profile in the ventral hippocampus during male pubertal development. (A–F) Fold change differences in mature miR expression levels at PND 30, 44, and 74 as analyzed by ^ΔΔCt method. Data are depicted as a scatter plot for each animal in each group, overlaid with a line of best fit. Dissimilar lower-case letters indicate statistically significant difference between groups. (G) Raw qPCR Ct values of mature miRs demonstrating relative abundance in the ventral hippocampus across pubertal development. Data are depicted as mean ± SEM. N = 10/age group Ct = cycle threshold.