CB1-mediated signaling in developmental ethanol effects

项目摘要 在怀孕期间，酒精滥用会在胎儿大脑中产生持续的变化， 一生的损伤。这些条件被定义为胎儿酒精谱系障碍（FASD）。最 令人关切的是，怀孕期间经常酗酒会导致后代的认知和社会行为缺陷。 然而，持续性酒精诱导的神经行为障碍的分子机制并不明确。 完全理解我们以前的研究表明，酒精暴露导致CB 1信号缺陷， 新生小鼠（P7），导致成年小鼠神经变性和行为缺陷。其他数据也表明CB 1 酒精的致畸作用然而，突触和行为缺陷是否是由于酒精诱导的 成年人海马（HP）和前额叶皮质（PFC）区域内CB 1及其信号事件的变化 仍然是未知的。CB 1主要表达于谷氨酸能和GABA能神经元。他们可以引出 细胞类型特异性信号传导取决于它们被激活的位置，并有助于神经元和行为 成果。CB 1活性也通过表观遗传学调节基因表达;然而，其机制尚不清楚。 我们最近的初步研究结果表明，新生小鼠中相当于人类妊娠晚期的酒精暴露会导致 持续的认知和社会行为缺陷。这些行为变化伴随着CB 1增加， 表达，组蛋白甲基化，以及突触结构和功能所必需的基因表达减少， 特定的神经元类型。这些发现表明酒精改变了基因表达的转录控制 和突触功能。这一提议将检验一种机制 PFC和HP中导致行为缺陷的潜在突触功能障碍是CB 1活性增强， 出生后酒精引起的组蛋白甲基化导致突触基因表达异常。这份提案将检验这些 使用细胞类型特异性（Cre或CreERT 2）RiboTag（TRAP）技术研究CB 1和 出生后酒精暴露（PAE）改变组蛋白甲基化事件。我们会特别注意证据 与脊柱和突触囊泡功能相关的组蛋白甲基化反应基因的异常调节。 此外，我们还将通过条件性免疫荧光技术研究组蛋白甲基化对突触结构和功能的作用。 在本发明的实施方案中，所述方法包括在所述细胞系中CB 1基因或编码组蛋白甲基化相关酶的基因（FloxP系）的缺失（cKO）。 在成人PFC和HP中特异性神经元类型（CreERT 2）。最后，我们将机械地测试 使用CB 1的cKO或特定神经元中的组蛋白甲基化酶功能调节组蛋白甲基化水平 拯救成年人的认知和社会行为。这些研究将使用性别依赖模型来执行一个 结合表观遗传学、突触和行为分析对PAE的反应。我们在本提案中的目标是 确定CB 1和组蛋白甲基化相关转录长期变化的机制 以及PFC和HP的突触变化，并确定其对持续认知和社会性的贡献。 由PAE引起的行为缺陷。这些发现将阐明途径，并提供可能的新靶点， PAE相关神经系统疾病的治疗干预。

Project Summary During pregnancy, alcohol abuse produces persistent changes in the fetal brain, causing behavioral impairments throughout life. These conditions are defined as fetal alcohol spectrum disorder (FASD). Most concerningly, regular binge drinking during pregnancy causes cognitive and socio-behavioral deficits in offspring. However, the molecular mechanisms underlying persistent alcohol-induced neurobehavioral impairments are not fully understood. Our previous studies demonstrated that alcohol exposure results in CB1 signaling defects in neonatal mice (P7) that cause neurodegeneration and behavioral deficits in adults. Other data also implicate CB1 in the teratogenic effects of alcohol. However, whether synaptic and behavioral deficits are due to alcohol-induced changes in CB1 and its signaling events within the adult hippocampus (HP) and prefrontal cortical (PFC) regions remains largely unknown. CB1s are mainly expressed in glutamatergic and GABAergic neurons. They can elicit cell-type-specific signaling depending on where they are activated and contribute to neuronal and behavior outcomes. CB1 activity also regulates gene expression via epigenetics; however, the mechanisms are unknown. Our recent pilot findings suggested that human third-trimester-equivalent alcohol exposure in neonatal mice caused persistent cognitive and social behavior deficits. These behavioral changes were accompanied by increased CB1 expression, histone methylation, and reduced expression of genes essential for synaptic structure and function in specific neuronal types. These findings suggest that alcohol alters the transcriptional control of gene expression and synaptic function in a given cell type in the PFC and HP. This proposal will test the hypothesis that a mechanism underlying synaptic dysfunction in the PFC and HP contributing to behavioral deficits is enhanced CB1 activity and histone methylation by postnatal alcohol leading to aberrant synaptic gene expression. This proposal will test these hypotheses in PFC and HP using cell-type-specific (Cre or CreERT2) RiboTag (TRAP) technology to study CB1 and histone methylation events altered by postnatal alcohol exposure (PAE). We will pay special attention to evidence of aberrant regulation of histone methylation responsive genes related to spine and synaptic vesicle function. Additionally, we will investigate the role of histone methylation on synaptic structure and function by conditional deletion (cKO) of the CB1 gene or a gene encoding a histone-methylation-related enzyme (FloxP lines) in the specific neuronal type (CreERT2) in the adult PFC and HP. Finally, we will mechanistically test the effects of modulating histone methylation levels using cKO of CB1 or histone methylation enzyme function in specific neuronal types to rescue adult cognitive and social behaviors. These studies will use sex-dependent models to perform a combined epigenetic, synaptic, and behavioral analysis of the response to PAE. Our goals in this proposal are to identify the mechanisms underlying the long-lasting changes in CB1 and histone-methylation-related transcriptional and synaptic changes in the PFC and HP and determine their contributions to the persistent cognitive and socio- behavioral deficits resulting from PAE. The findings will elucidate pathways and provide possible novel targets for therapeutic intervention in PAE-related neurological disease.