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Ethanol exposure induces neonatal neurodegeneration by enhancing CB1R Exon1 histone H4K8 acetylation and up-regulating CB1R function causing neurobehavioral abnormalities in adult mice.

Subbanna S, Nagre NN, Umapathy NS, Pace BS, Basavarajappa BS - Int. J. Neuropsychopharmacol. (2014)

Bottom Line: We found that ethanol treatment of P7 mice enhances acetylation of H4 on lysine 8 (H4K8ace) at CB1R exon1, CB1R binding as well as the CB1R agonist-stimulated GTPγS binding in the hippocampus and neocortex, two brain regions that are vulnerable to ethanol at P7 and are important for memory formation and storage, respectively.We also found that ethanol inhibits cyclic adenosine monophosphate response element-binding protein (CREB) phosphorylation and activity-regulated cytoskeleton-associated protein (Arc) expression in neonatal and adult mice.The blockade or genetic deletion of CB1Rs prior to ethanol treatment at P7 rescued CREB phosphorylation and Arc expression.

View Article: PubMed Central - PubMed

Affiliation: Division of Analytical Psychopharmacology, Nathan Kline Institute for Psychiatric Research, Orangeburg, NY (Drs Subbanna, Nagre, and Basavarajappa); Vascular Biology Center, Georgia Regents University, Augusta, GA (Dr Umapathy); Department of Pediatrics, Georgia Regents University, Augusta, GA (Dr Pace); New York State Psychiatric Institute, New York, NY (Dr Basavarajappa); Department of Psychiatry, College of Physicians & Surgeons, Columbia University, New York, NY (Dr Basavarajappa).

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Pharmacological blockade or genetic ablation of CB1Rs provides protection against ethanol-induced inhibition of CREB phosphorylation in the neonatal mouse brain. (a) Hippocampal and neocortical nuclear extracts from the four treatment groups (S+V, E+V, S+SR, and E+ SR) were subjected to Western blot to analyze the levels of pCREB and CREB (n = 10 pups/group; ***p < 0.001 vs. S+V; #p < 0.001 vs. E+V). (b) Additional Western blot analyses were performed to determine the levels of pCREB and CREB in the hippocampal and cortical nuclear extracts obtained from the saline and ethanol-treated P7 CB1RWT and KO mice. The representative blots are shown for the hippocampal and cortical nuclear extracts (n = 10 pups/group; ***p < 0.001 vs. S+CB1RWT; #p < 0.001 vs. E+ CB1RWT; @p < 0.001 vs. S+CB1RWT). β-actin was used as a loading control. Two-way ANOVA with Bonferroni’s post hoc tests was used for statistical analysis. Each point is the mean ± SEM. HP, hippocampus; NC, neocortex.
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Figure 3: Pharmacological blockade or genetic ablation of CB1Rs provides protection against ethanol-induced inhibition of CREB phosphorylation in the neonatal mouse brain. (a) Hippocampal and neocortical nuclear extracts from the four treatment groups (S+V, E+V, S+SR, and E+ SR) were subjected to Western blot to analyze the levels of pCREB and CREB (n = 10 pups/group; ***p < 0.001 vs. S+V; #p < 0.001 vs. E+V). (b) Additional Western blot analyses were performed to determine the levels of pCREB and CREB in the hippocampal and cortical nuclear extracts obtained from the saline and ethanol-treated P7 CB1RWT and KO mice. The representative blots are shown for the hippocampal and cortical nuclear extracts (n = 10 pups/group; ***p < 0.001 vs. S+CB1RWT; #p < 0.001 vs. E+ CB1RWT; @p < 0.001 vs. S+CB1RWT). β-actin was used as a loading control. Two-way ANOVA with Bonferroni’s post hoc tests was used for statistical analysis. Each point is the mean ± SEM. HP, hippocampus; NC, neocortex.

Mentions: To elucidate the downstream intracellular pathways involved in the protective effects of the CB1R blockade, we studied the involvement of the pCREB and Arc pathway, a key regulator of cell survival (Luikart et al., 2008) and synaptic plasticity (Caroni et al., 2012). We investigated whether pre-treatment of SR, which prevents ethanol-induced neurodegeneration, could rescue these ethanol-induced pCREB and Arc deficits. Our results suggest that CREB phosphorylation, as well as Arc protein expression, were rescued by SR pre-treatment (compared with the ethanol group) in neonatal (HP: pCREB, F3, 20 = 55, p > 0.01, Arc, F3, 20 = 30, p > 0.01; NC: pCREB, F3, 20 = 35, p > 0.01, Arc, F3, 20 = 45, p > 0.01, two-way ANOVA; Figure 3a and 4a) and adult mice tissues (HP: pCREB, F3, 20 = 40, p > 0.01, Arc, F3, 20 = 25, p > 0.01; NC: pCREB, F3, 20 = 45, p > 0.01, Arc, F3, 20 = 55, p > 0.01, two-way ANOVA; Figure 5a and 6a). We found that the total CREB protein levels were not altered in the ethanol-treated samples compared with the saline samples. In addition, SR did not alter the CREB protein levels in either the ethanol or saline samples of neonatal (Figure 3a) and adult (Figure 5a) rats. Similarly, CB1RKO mice, which do not exhibit ethanol-induced neurodegeneration, provided protection against P7 ethanol-induced inhibition of CREB phosphorylation and Arc expression in the neonatal (HP: pCREB, F3, 20 = 42, p < 0.001, Arc, F3, 20 = 52, p < 0.001; NC: pCREB, F3, 20 = 58, p < 0.001, Arc, F3, 20 = 62, p < 0.001, one-way ANOVA; Figure 3b and 4b) and adult mice tissues (HP: pCREB, F3, 20 = 22, p < 0.01, Arc, F3, 20 = 32, p < 0.01; NC: pCREB, F3, 20 = 38, p < 0.01, Arc, F3, 20 = 32, p < 0.01, one-way ANOVA; Figure 5b and 6b). In addition, neonatal and adult CB1RKO mice also exhibited enhanced CREB phosphorylation and Arc levels compared to their WT littermates (p < 0.01; Figure 5b and 6b).


Ethanol exposure induces neonatal neurodegeneration by enhancing CB1R Exon1 histone H4K8 acetylation and up-regulating CB1R function causing neurobehavioral abnormalities in adult mice.

Subbanna S, Nagre NN, Umapathy NS, Pace BS, Basavarajappa BS - Int. J. Neuropsychopharmacol. (2014)

Pharmacological blockade or genetic ablation of CB1Rs provides protection against ethanol-induced inhibition of CREB phosphorylation in the neonatal mouse brain. (a) Hippocampal and neocortical nuclear extracts from the four treatment groups (S+V, E+V, S+SR, and E+ SR) were subjected to Western blot to analyze the levels of pCREB and CREB (n = 10 pups/group; ***p < 0.001 vs. S+V; #p < 0.001 vs. E+V). (b) Additional Western blot analyses were performed to determine the levels of pCREB and CREB in the hippocampal and cortical nuclear extracts obtained from the saline and ethanol-treated P7 CB1RWT and KO mice. The representative blots are shown for the hippocampal and cortical nuclear extracts (n = 10 pups/group; ***p < 0.001 vs. S+CB1RWT; #p < 0.001 vs. E+ CB1RWT; @p < 0.001 vs. S+CB1RWT). β-actin was used as a loading control. Two-way ANOVA with Bonferroni’s post hoc tests was used for statistical analysis. Each point is the mean ± SEM. HP, hippocampus; NC, neocortex.
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Figure 3: Pharmacological blockade or genetic ablation of CB1Rs provides protection against ethanol-induced inhibition of CREB phosphorylation in the neonatal mouse brain. (a) Hippocampal and neocortical nuclear extracts from the four treatment groups (S+V, E+V, S+SR, and E+ SR) were subjected to Western blot to analyze the levels of pCREB and CREB (n = 10 pups/group; ***p < 0.001 vs. S+V; #p < 0.001 vs. E+V). (b) Additional Western blot analyses were performed to determine the levels of pCREB and CREB in the hippocampal and cortical nuclear extracts obtained from the saline and ethanol-treated P7 CB1RWT and KO mice. The representative blots are shown for the hippocampal and cortical nuclear extracts (n = 10 pups/group; ***p < 0.001 vs. S+CB1RWT; #p < 0.001 vs. E+ CB1RWT; @p < 0.001 vs. S+CB1RWT). β-actin was used as a loading control. Two-way ANOVA with Bonferroni’s post hoc tests was used for statistical analysis. Each point is the mean ± SEM. HP, hippocampus; NC, neocortex.
Mentions: To elucidate the downstream intracellular pathways involved in the protective effects of the CB1R blockade, we studied the involvement of the pCREB and Arc pathway, a key regulator of cell survival (Luikart et al., 2008) and synaptic plasticity (Caroni et al., 2012). We investigated whether pre-treatment of SR, which prevents ethanol-induced neurodegeneration, could rescue these ethanol-induced pCREB and Arc deficits. Our results suggest that CREB phosphorylation, as well as Arc protein expression, were rescued by SR pre-treatment (compared with the ethanol group) in neonatal (HP: pCREB, F3, 20 = 55, p > 0.01, Arc, F3, 20 = 30, p > 0.01; NC: pCREB, F3, 20 = 35, p > 0.01, Arc, F3, 20 = 45, p > 0.01, two-way ANOVA; Figure 3a and 4a) and adult mice tissues (HP: pCREB, F3, 20 = 40, p > 0.01, Arc, F3, 20 = 25, p > 0.01; NC: pCREB, F3, 20 = 45, p > 0.01, Arc, F3, 20 = 55, p > 0.01, two-way ANOVA; Figure 5a and 6a). We found that the total CREB protein levels were not altered in the ethanol-treated samples compared with the saline samples. In addition, SR did not alter the CREB protein levels in either the ethanol or saline samples of neonatal (Figure 3a) and adult (Figure 5a) rats. Similarly, CB1RKO mice, which do not exhibit ethanol-induced neurodegeneration, provided protection against P7 ethanol-induced inhibition of CREB phosphorylation and Arc expression in the neonatal (HP: pCREB, F3, 20 = 42, p < 0.001, Arc, F3, 20 = 52, p < 0.001; NC: pCREB, F3, 20 = 58, p < 0.001, Arc, F3, 20 = 62, p < 0.001, one-way ANOVA; Figure 3b and 4b) and adult mice tissues (HP: pCREB, F3, 20 = 22, p < 0.01, Arc, F3, 20 = 32, p < 0.01; NC: pCREB, F3, 20 = 38, p < 0.01, Arc, F3, 20 = 32, p < 0.01, one-way ANOVA; Figure 5b and 6b). In addition, neonatal and adult CB1RKO mice also exhibited enhanced CREB phosphorylation and Arc levels compared to their WT littermates (p < 0.01; Figure 5b and 6b).

Bottom Line: We found that ethanol treatment of P7 mice enhances acetylation of H4 on lysine 8 (H4K8ace) at CB1R exon1, CB1R binding as well as the CB1R agonist-stimulated GTPγS binding in the hippocampus and neocortex, two brain regions that are vulnerable to ethanol at P7 and are important for memory formation and storage, respectively.We also found that ethanol inhibits cyclic adenosine monophosphate response element-binding protein (CREB) phosphorylation and activity-regulated cytoskeleton-associated protein (Arc) expression in neonatal and adult mice.The blockade or genetic deletion of CB1Rs prior to ethanol treatment at P7 rescued CREB phosphorylation and Arc expression.

View Article: PubMed Central - PubMed

Affiliation: Division of Analytical Psychopharmacology, Nathan Kline Institute for Psychiatric Research, Orangeburg, NY (Drs Subbanna, Nagre, and Basavarajappa); Vascular Biology Center, Georgia Regents University, Augusta, GA (Dr Umapathy); Department of Pediatrics, Georgia Regents University, Augusta, GA (Dr Pace); New York State Psychiatric Institute, New York, NY (Dr Basavarajappa); Department of Psychiatry, College of Physicians & Surgeons, Columbia University, New York, NY (Dr Basavarajappa).

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Related in: MedlinePlus