Ethanol Exposure Induces Neonatal Neurodegeneration by Enhancing CB1R Exon1 Histone H4K8 Acetylation and Up-regulating CB1R Function causing Neurobehavioral Abnormalities in Adult Mice

Abstract

Background:Ethanol exposure to rodents during postnatal day 7 (P7), which is comparable to the third trimester of human pregnancy, induces long-term potentiation and memory deficits. However, the molecular mechanisms underlying these deficits are still poorly understood.Methods:In the present study, we explored the potential role of epigenetic changes at cannabinoid type 1 (CB1R) exon1 and additional CB1R functions, which could promote memory deficits in animal models of fetal alcohol spectrum disorder.Results: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. CB1R knockout mice exhibited neither ethanol-induced neurodegeneration nor inhibition of CREB phosphorylation or Arc expression. However, both neonatal and adult mice did exhibit enhanced CREB phosphorylation and Arc protein expression. P7 ethanol-treated adult mice exhibited impaired spatial and social recognition memory, which were prevented by the pharmacological blockade or deletion of CB1Rs at P7.Conclusions:Together, these findings suggest that P7 ethanol treatment induces CB1R expression through epigenetic modification of the CB1R gene, and that the enhanced CB1R function induces pCREB, Arc, spatial, and social memory deficits in adult mice.

Distinct functions of endogenous cannabinoid system in alcohol abuse disorders

“…In addition to increased AEA and associated biosynthetic enzymes, the alcohol‐induced transcriptional activation of the Cnr1 gene results in increased levels of Cnr1 mRNA and CB 1 receptor protein expression in cortical and hippocampal brain regions (Subbanna, Shivakumar, Psychoyos, Xie, & Basavarajappa, ). Remarkably, we found that postnatal alcohol exposure in mice enhances the acetylation of histone (H4) on Lys 8 (H4K8ace) in exon 1 of Cnr1 and CB 1 receptor binding and CB 1 receptor agonist‐stimulated GTPγS binding in cortical and hippocampal brain regions (Subbanna, Nagre, Umapathy, Pace, & Basavarajappa, ). The administration of SR or the genetic ablation of CB 1 receptors (CB 1 −/− ) before alcohol exposure prevents neuronal cell death (Subbanna et al, ; Subbanna et al, ).…”

A single day of 5-azacytidine exposure during development induces neurodegeneration in neonatal mice and neurobehavioral deficits in adult mice

“…Genetic deletion of Tet1 impairs several synaptic plasticity-related genes, including Arc, and induces abnormal hippocampal synaptic plasticity and impaired spatial [116] and contextual fear memory [117]. Although future studies are required to understand the brain region specific epigenome changes and their relationship to observed neurobehavioral deficits in adult animals, our studies clearly suggest that the impairment of DNA methylation during early brain development affects the expression of survival factors [118] such as Arc [23] expression, inducing a delay in neuronal maturation [119]. Moreover, such impairment may be responsible for the observed object, spatial and social recognition memory deficits and synaptic plasticity abnormalities in adult mice.…”

“…Our findings suggest that the mechanism by which 5-AzaC induces caspase-3 activation differs significantly from that of alcohol-induced caspase-3 activation in P7 mice [9]. This is partly because pre-administration of a G9a/GLP inhibitor (Bix) [22, 35] or CB1R antagonist (SR) [19, 23, 24], which are known to prevent alcohol-induced caspase-3 activation, failed to rescue 5-AzaC-induced caspase-3 activation in neonatal mice. Furthermore, the CB1RKO, which provides protection against alcohol-induced caspase-3 activation [19, 23, 24], also failed to rescue 5-AzaC-induced caspase-3 activation in neonatal mice.…”

“…This is partly because pre-administration of a G9a/GLP inhibitor (Bix) [22, 35] or CB1R antagonist (SR) [19, 23, 24], which are known to prevent alcohol-induced caspase-3 activation, failed to rescue 5-AzaC-induced caspase-3 activation in neonatal mice. Furthermore, the CB1RKO, which provides protection against alcohol-induced caspase-3 activation [19, 23, 24], also failed to rescue 5-AzaC-induced caspase-3 activation in neonatal mice. Although future studies are required to identify the DNA hypomethylation-induced regulation of apoptosis-related genes in P7 mice, it should be noted that the DNA methylation inhibitor activity of 5-AzaC involves its incorporation into cellular DNA/RNA, with subsequent sequestration of DNA methyltransferases (DNMTs) through the formation of a covalent bond between C6 of 5-AzaC and the cysteine thiolate of DNMTs [62].…”

“…The social recognition memory procedure [44, 45] allows direct access between the experimental and the stimulus mice and is widely used to assess long-term memory in rodents [22, 23, 44, 46, 47]. Briefly, three-month-old male mice that had been treated with saline or 5-AzaC at P7 (n = 8 mice from four different litters/group) were subjected to a social recognition memory test that was performed as described previously [22].…”