Adolescent alcohol exposure alters lysine demethylase 1 (LSD1) expression and histone methylation in the amygdala during adulthood - PubMed (original) (raw)

. 2017 Sep;22(5):1191-1204.

doi: 10.1111/adb.12404. Epub 2016 May 15.

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Adolescent alcohol exposure alters lysine demethylase 1 (LSD1) expression and histone methylation in the amygdala during adulthood

Evan J Kyzar et al. Addict Biol. 2017 Sep.

Abstract

Alcohol exposure in adolescence is an important risk factor for the development of alcoholism in adulthood. Epigenetic processes are implicated in the persistence of adolescent alcohol exposure-related changes, specifically in the amygdala. We investigated the role of histone methylation mechanisms in the persistent effects of adolescent intermittent ethanol (AIE) exposure in adulthood. Adolescent rats were exposed to 2 g/kg ethanol (2 days on/off) or intermittent n-saline (AIS) during postnatal days (PND) 28-41 and used for behavioral and epigenetic studies. We found that AIE exposure caused a long-lasting decrease in mRNA and protein levels of lysine demethylase 1(Lsd1) and mRNA levels of Lsd1 + 8a (a neuron-specific splice variant) in specific amygdaloid structures compared with AIS-exposed rats when measured at adulthood. Interestingly, AIE increased histone H3 lysine 9 dimethylation (H3K9me2) levels in the central nucleus of the amygdala (CeA) and medial nucleus of the amygdala (MeA) in adulthood without producing any change in H3K4me2 protein levels. Acute ethanol challenge (2 g/kg) in adulthood attenuated anxiety-like behaviors and the decrease in Lsd1 + 8a mRNA levels in the amygdala induced by AIE. AIE caused an increase in H3K9me2 occupancy at the brain-derived neurotrophic factor exon IV promoter in the amygdala that returned to baseline after acute ethanol challenge in adulthood. These results indicate that AIE specifically modulates epizymes involved in H3K9 dimethylation in the amygdala in adulthood, which are possibly responsible for AIE-induced chromatin remodeling and adult psychopathology such as anxiety.

Keywords: alcohol; amygdala; anxiety; epigenetics; histone demethylase; histone methylation; lysine demethylase 1.

© Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

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Figures

Figure 1

Figure 1. mRNA profiling of histone methyltransferases and demethylases in the amygdala (A) and BNST (B) of adolescent rats exposed to AIE

Rats were exposed to adolescent intermittent ethanol (AIE) or saline (AIS) and their brains were collected and amygdaloid as well as BNST tissues were dissected out for mRNA analysis at postnatal day (PND) 41-42 either 1 hr after last ethanol exposure (AIE group) or 24 hrs after last ethanol exposure during withdrawal (AIW group) in the amygdala (A) and BNST (B). Values are represented as mean ± SEM of n=6-7 rats and are significantly different from AIS-exposed controls (*p < 0.05, **p<0.01, one-way ANOVA followed by post hoc Tukey's test).

Figure 2

Figure 2. mRNA profiling of histone methyltransferases and demethylases in the amygdala (A) and BNST (B) of adult rats previously exposed to AIE

Rats were exposed to adolescent intermittent ethanol (AIE) or saline (AIS) and allowed to grow to adulthood without further ethanol or saline exposure. Their brains were collected and amygdaloid as well as BNST tissues were dissected out for mRNA analysis at postnatal day (PND) 92. Values are represented as mean ± SEM of n=5-8 rats and are significantly different from AIS-exposed group (*p < 0.05, Student's t test).

Figure 3

Figure 3. Decreased LSD1 protein and Lsd1+8a mRNA in specific amygdaloid structures of adult rats exposed to AIE

A) Photomicrographs (Scale bar = 50 μm) showing the gold immunolabeling of LSD1 in the central (CeA) and medial nucleus of amygdala (MeA) of adolescent intermittent ethanol (AIE) - or saline (AIS)-exposed adult rats. B) Quantification of LSD1 protein levels by gold immunolabeling in the CeA, MeA and basolateral amygdala (BLA) of AIS- and AIE-exposed adult rats. C) Photomicrographs (Scale bar = 50 μm) showing the Lsd1+8a mRNA positive cells (in-situ PCR) in the CeA and MeA of AIE- and AIS-exposed adult rats. D) Quantification of Lsd1+8a mRNA levels by in situ PCR in the CeA, MeA and BLA of AIS- and AIE-exposed adult rats. Values are represented as mean ± SEM of n=5 rats and are significantly different from AIS-exposed group (**p < 0.01, ***p < 0.001, Student's t test).

Figure 4

Figure 4. Increased histone H3 lysine 9 dimethlyation (H3K9me2) but no change in H3K4me2 protein levels in the amygdala of AIE adult rats

A) Photomicrographs (Scale bar = 50 μm) showing the gold immunolabeling of H3K4me2 proteins in the central (CeA) and medial nucleus of amygdala (MeA) of adolescent intermittent ethanol (AIE)- or saline (AIS)-exposed adult rats. B) Quantification of H3K4me2 protein levels by gold immunolabeling in the CeA, MeA and basolateral amygdala (BLA) of AIS- and AIE-exposed adult rats. C) Photomicrographs (Scale bar = 50 μm) showing the gold immunolabeling of H3K9me2 proteins in the CeA and MeA of AIE- and AIS-exposed adult rats. D) Quantification of H3K9me2 protein levels by gold immunolabeling in the CeA, MeA and BLA of AIS- and AIE-exposed adult rats. Values are represented as mean ± SEM of n=5 rats and are significantly different from AIS-exposed group (***p < 0.001, Student's t test). E) Representative confocal photomicrographs of immunefluorescent staining (Scale bar = 50 μm) of H3K9me2 (red) co-localized with either neuron-specific nuclear protein (NeuN; green) or glial fibrillary acidic protein (GFAP; green) in the CeA and MeA of naive adult rats.

Figure 5

Figure 5. mRNA profiling of (A) _Lsd1_-interacting partners and (B) histone H3 lysine 9 (H3K9) methylation modifying enzymes reveals a specific decrease in Kdm4c in the amygdala of AIE adult rats

Rats were exposed to adolescent intermittent ethanol (AIE) or saline (AIS) and allowed to grow to adulthood without further ethanol exposure. Their brains were collected and amygdaloid tissues dissected out for mRNA analysis at postnatal day (PND) 92. Values are represented as mean ± SEM of n=5-9 rats and are significantly different from AIS-exposed group (*p < 0.05, Student's t test). AR, androgen receptor; TLX, nuclear receptor.

Figure 6

Figure 6. Acute ethanol challenge normalizes (A) anxiety-like behavior, (B) amygdala Lsd1+8a expression, and (C) amygdala histone H3 lysine 9 dimethylation (H3K9me2) occupancy at Bdnf exon IV promoter

Rats were exposed to adolescent intermittent ethanol (AIE) or saline (AIS) in adolescence (postnatal day [PND] 28-41) and no further exposure to ethanol until animals were exposed to an acute ethanol challenge (2 g/kg) at PND 101 or 102. Animals were tested in the elevated plus maze (EPM) for anxiety-like behaviors (activities in open and closed arms), and immediately brains were collected and amygdaloid tissues dissected out for mRNA and chromatin immunoprecipitation (ChIP) analysis. Values are represented as mean ± SEM of n=6-8 rats and are significantly different between groups (*p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA followed by post hoc Tukey's test).

Figure 7

Figure 7

Hypothetical model showing adolescent intermittent ethanol (AIE) exposure causes a lasting decrease in lysine demethylase 1(LSD1) in the central nucleus of amygdala (CeA) and a decrease in Lsd1+8a in the CeA and medial nucleus of amygdala (MeA) in adulthood. Adolescent intermittent ethanol (AIE)-exposed adults show increased histone H3 lysine 9 dimethylation (H3K9me2) in the CeA and MeA with no change in histone H3 lysine 4 dimethylation (H3K4me2) levels. In addition, AIE increased the levels of H3K9me2 at the promoter of Bdnf exon IV in the amygdala. This, along with previous data from our lab showing an increase in histone deacetylase isoform 2 (HDAC2) and the resulting decrease in global and Bdnf exon IV-specific H3K9 acetylation and associated reduced levels of BDNF in the amygdala (Pandey et al., 2015) likely contributes to condensed chromatin architecture and increased anxiety-like and alcohol-drinking behaviors.

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