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Alcohol exposure decreases CREB binding protein expression and histone acetylation in the developing cerebellum.

Guo W, Crossey EL, Zhang L, Zucca S, George OL, Valenzuela CF, Zhao X - PLoS ONE (2011)

Bottom Line: However, the underlying molecular mechanism is unclear.CBP is a histone acetyltransferase, a component of the epigenetic mechanism controlling neuronal gene expression.This effect of ethanol may be responsible for the motor coordination deficits that characterize fetal alcohol spectrum disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences, University of New Mexico School of Medicine, Albuquerque, New Mexico, United States of America.

ABSTRACT

Background: Fetal alcohol exposure affects 1 in 100 children making it the leading cause of mental retardation in the US. It has long been known that alcohol affects cerebellum development and function. However, the underlying molecular mechanism is unclear.

Methodology/principal findings: We demonstrate that CREB binding protein (CBP) is widely expressed in granule and Purkinje neurons of the developing cerebellar cortex of naïve rats. We also show that exposure to ethanol during the 3(rd) trimester-equivalent of human pregnancy reduces CBP levels. CBP is a histone acetyltransferase, a component of the epigenetic mechanism controlling neuronal gene expression. We further demonstrate that the acetylation of both histone H3 and H4 is reduced in the cerebellum of ethanol-treated rats.

Conclusions/significance: These findings indicate that ethanol exposure decreases the expression and function of CBP in the developing cerebellum. This effect of ethanol may be responsible for the motor coordination deficits that characterize fetal alcohol spectrum disorders.

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Related in: MedlinePlus

AcH3 expression was decreased in the cerebellum of ethanol-exposed developing rats.(A) Image of one of the western blots showing differential acetylated H3 (AcH3) expression in the cerebellum isolated from control pups (exposed to air) analyzed at P2 to P12. (B) Representative AcH3 western blot analysis of cerebellum isolated from ethanol-exposed pups analyzed at P2 to P12. (C) Quantitative analysis showing that AcH3 protein levels (normalized to β-actin) are lower in the ethanol group compared to the control group between P2 and P10, but not at P12. Three independently exposed liters were analyzed and each was counted as an n = 1. Multiple western blots were analyzed for each exposure to minimize the variation between western blots. (D) Representative total H3 (Total H3) western blot analysis of cerebellum isolated from control pups (exposed to air) analyzed at P2 to P12. (E) Representative total H3 western blot analysis of cerebellum isolated from ethanol-exposed pups analyzed at P2 to P12. (F) Quantitative analysis showing that total H3 protein levels (normalized to β-actin) are not changed in the ethanol group compared to the control group. (G) Quantitative analysis showing that the ratio of AcH3 compared to total H3 protein levels are lower in the ethanol group compared to the control group between P2 and P10, but not at P12. (H) Representative AcH3 immunohistological images of control and ethanol exposed cerebella at P8. AcH3, red; Dapi, blue. Scale bar = 50 µm. (I) Quantitative analysis of AcH3 immunohistological signal intensity demonstrates that AcH3 expression is reduced in the cerebellum of ethanol exposed rats compared to controls. N = 4 individual animal/condition. P<0.05 t-test.
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pone-0019351-g005: AcH3 expression was decreased in the cerebellum of ethanol-exposed developing rats.(A) Image of one of the western blots showing differential acetylated H3 (AcH3) expression in the cerebellum isolated from control pups (exposed to air) analyzed at P2 to P12. (B) Representative AcH3 western blot analysis of cerebellum isolated from ethanol-exposed pups analyzed at P2 to P12. (C) Quantitative analysis showing that AcH3 protein levels (normalized to β-actin) are lower in the ethanol group compared to the control group between P2 and P10, but not at P12. Three independently exposed liters were analyzed and each was counted as an n = 1. Multiple western blots were analyzed for each exposure to minimize the variation between western blots. (D) Representative total H3 (Total H3) western blot analysis of cerebellum isolated from control pups (exposed to air) analyzed at P2 to P12. (E) Representative total H3 western blot analysis of cerebellum isolated from ethanol-exposed pups analyzed at P2 to P12. (F) Quantitative analysis showing that total H3 protein levels (normalized to β-actin) are not changed in the ethanol group compared to the control group. (G) Quantitative analysis showing that the ratio of AcH3 compared to total H3 protein levels are lower in the ethanol group compared to the control group between P2 and P10, but not at P12. (H) Representative AcH3 immunohistological images of control and ethanol exposed cerebella at P8. AcH3, red; Dapi, blue. Scale bar = 50 µm. (I) Quantitative analysis of AcH3 immunohistological signal intensity demonstrates that AcH3 expression is reduced in the cerebellum of ethanol exposed rats compared to controls. N = 4 individual animal/condition. P<0.05 t-test.

Mentions: Since CBP functions through its intrinsic HAT activity, we examined whether the decrease in CBP expression was associated with a reduction in histone acetylation levels. We chose to study histones type-3 (H3) and -4 (H4), as these are well known substrates of the HAT activity of CBP [37], [45] and CBP and AcH3 and AcH4 are colocalized in the developing cerebellum (Fig. S1). As shown in Fig. 5, in control brains, acetylated H3 levels were high at postnatal days 2–8 and decreased slightly during postnatal days 10–12 (Fig. 5A, C) while total H3 levels were constant (Fig. 5D, F) during this developmental period. Ethanol exposure significantly reduced acetylated H3 levels at postnatal days 2–10 (Fig. 5B, C), but had no significant effect on total H3 protein levels (Fig. 5E, F). Consequently, the levels of acetylated H3 normalized to total H3 protein were significantly reduced as a result of ethanol exposure from P2 through P10 (Fig. 5G). In addition, immunohistochemical studies revealed a decrease in acetylated H3 levels in cerebellar sections from the ethanol-exposed group at postnatal day 8 (Fig. 5H, I). To determine if the effect of ethanol exposure is specific for H3K9/K14 acetylation, we then used an antibody that specifically recognizes H3 acetylation at a different residue (K23) and found that the levels of acetyl-H3K23 were also reduced in alcohol-exposed group (Fig. S2).


Alcohol exposure decreases CREB binding protein expression and histone acetylation in the developing cerebellum.

Guo W, Crossey EL, Zhang L, Zucca S, George OL, Valenzuela CF, Zhao X - PLoS ONE (2011)

AcH3 expression was decreased in the cerebellum of ethanol-exposed developing rats.(A) Image of one of the western blots showing differential acetylated H3 (AcH3) expression in the cerebellum isolated from control pups (exposed to air) analyzed at P2 to P12. (B) Representative AcH3 western blot analysis of cerebellum isolated from ethanol-exposed pups analyzed at P2 to P12. (C) Quantitative analysis showing that AcH3 protein levels (normalized to β-actin) are lower in the ethanol group compared to the control group between P2 and P10, but not at P12. Three independently exposed liters were analyzed and each was counted as an n = 1. Multiple western blots were analyzed for each exposure to minimize the variation between western blots. (D) Representative total H3 (Total H3) western blot analysis of cerebellum isolated from control pups (exposed to air) analyzed at P2 to P12. (E) Representative total H3 western blot analysis of cerebellum isolated from ethanol-exposed pups analyzed at P2 to P12. (F) Quantitative analysis showing that total H3 protein levels (normalized to β-actin) are not changed in the ethanol group compared to the control group. (G) Quantitative analysis showing that the ratio of AcH3 compared to total H3 protein levels are lower in the ethanol group compared to the control group between P2 and P10, but not at P12. (H) Representative AcH3 immunohistological images of control and ethanol exposed cerebella at P8. AcH3, red; Dapi, blue. Scale bar = 50 µm. (I) Quantitative analysis of AcH3 immunohistological signal intensity demonstrates that AcH3 expression is reduced in the cerebellum of ethanol exposed rats compared to controls. N = 4 individual animal/condition. P<0.05 t-test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3104983&req=5

pone-0019351-g005: AcH3 expression was decreased in the cerebellum of ethanol-exposed developing rats.(A) Image of one of the western blots showing differential acetylated H3 (AcH3) expression in the cerebellum isolated from control pups (exposed to air) analyzed at P2 to P12. (B) Representative AcH3 western blot analysis of cerebellum isolated from ethanol-exposed pups analyzed at P2 to P12. (C) Quantitative analysis showing that AcH3 protein levels (normalized to β-actin) are lower in the ethanol group compared to the control group between P2 and P10, but not at P12. Three independently exposed liters were analyzed and each was counted as an n = 1. Multiple western blots were analyzed for each exposure to minimize the variation between western blots. (D) Representative total H3 (Total H3) western blot analysis of cerebellum isolated from control pups (exposed to air) analyzed at P2 to P12. (E) Representative total H3 western blot analysis of cerebellum isolated from ethanol-exposed pups analyzed at P2 to P12. (F) Quantitative analysis showing that total H3 protein levels (normalized to β-actin) are not changed in the ethanol group compared to the control group. (G) Quantitative analysis showing that the ratio of AcH3 compared to total H3 protein levels are lower in the ethanol group compared to the control group between P2 and P10, but not at P12. (H) Representative AcH3 immunohistological images of control and ethanol exposed cerebella at P8. AcH3, red; Dapi, blue. Scale bar = 50 µm. (I) Quantitative analysis of AcH3 immunohistological signal intensity demonstrates that AcH3 expression is reduced in the cerebellum of ethanol exposed rats compared to controls. N = 4 individual animal/condition. P<0.05 t-test.
Mentions: Since CBP functions through its intrinsic HAT activity, we examined whether the decrease in CBP expression was associated with a reduction in histone acetylation levels. We chose to study histones type-3 (H3) and -4 (H4), as these are well known substrates of the HAT activity of CBP [37], [45] and CBP and AcH3 and AcH4 are colocalized in the developing cerebellum (Fig. S1). As shown in Fig. 5, in control brains, acetylated H3 levels were high at postnatal days 2–8 and decreased slightly during postnatal days 10–12 (Fig. 5A, C) while total H3 levels were constant (Fig. 5D, F) during this developmental period. Ethanol exposure significantly reduced acetylated H3 levels at postnatal days 2–10 (Fig. 5B, C), but had no significant effect on total H3 protein levels (Fig. 5E, F). Consequently, the levels of acetylated H3 normalized to total H3 protein were significantly reduced as a result of ethanol exposure from P2 through P10 (Fig. 5G). In addition, immunohistochemical studies revealed a decrease in acetylated H3 levels in cerebellar sections from the ethanol-exposed group at postnatal day 8 (Fig. 5H, I). To determine if the effect of ethanol exposure is specific for H3K9/K14 acetylation, we then used an antibody that specifically recognizes H3 acetylation at a different residue (K23) and found that the levels of acetyl-H3K23 were also reduced in alcohol-exposed group (Fig. S2).

Bottom Line: However, the underlying molecular mechanism is unclear.CBP is a histone acetyltransferase, a component of the epigenetic mechanism controlling neuronal gene expression.This effect of ethanol may be responsible for the motor coordination deficits that characterize fetal alcohol spectrum disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences, University of New Mexico School of Medicine, Albuquerque, New Mexico, United States of America.

ABSTRACT

Background: Fetal alcohol exposure affects 1 in 100 children making it the leading cause of mental retardation in the US. It has long been known that alcohol affects cerebellum development and function. However, the underlying molecular mechanism is unclear.

Methodology/principal findings: We demonstrate that CREB binding protein (CBP) is widely expressed in granule and Purkinje neurons of the developing cerebellar cortex of naïve rats. We also show that exposure to ethanol during the 3(rd) trimester-equivalent of human pregnancy reduces CBP levels. CBP is a histone acetyltransferase, a component of the epigenetic mechanism controlling neuronal gene expression. We further demonstrate that the acetylation of both histone H3 and H4 is reduced in the cerebellum of ethanol-treated rats.

Conclusions/significance: These findings indicate that ethanol exposure decreases the expression and function of CBP in the developing cerebellum. This effect of ethanol may be responsible for the motor coordination deficits that characterize fetal alcohol spectrum disorders.

Show MeSH
Related in: MedlinePlus