Prenatal Cannabis Exposure and Epigenetic Mechanisms Underlying Vulnerability to

描述（由申请人提供）：越来越多的州正在将大麻（大麻sativa）合法化，但其神经生物学后果在很大程度上是未知的。在怀孕期间，人类胎儿特别容易受到外源性损伤，例如暴露于药物。迄今为止，大麻是北美孕妇最常滥用的非法药物。流行病学研究证明，早期反复接触大麻与随后出现神经精神异常（包括吸毒成瘾和精神分裂障碍）的风险增加之间存在显著联系。众所周知，大麻以大麻素受体为靶点，大麻素受体在产前参与大脑的硬连接，并在控制伏隔核（NAc）多巴胺神经传递的神经元上表达。来自我们独特的人类胎儿大脑收集和大鼠模型的数据显示，产前大麻/四氢大麻酚暴露后，NAc中多巴胺能D2受体（Drd2）基因表达显著降低。有趣的是，我们的动物研究表明，这种D2受体的减少是由mRNA表达的改变引起的，并持续到成年期，从而表明了一种分子机制，这种机制可以维持长期的时间效应，而不能用个体基因型来解释。表观基因组控制基因调控并受环境影响，因此它是一个高度相关的生物学候选物，能够维持由于发育性药物暴露而导致的D2受体功能异常。已知对神经发育至关重要的主要表观遗传机制是染色质上核小体组蛋白的共价修饰，其调节基因转录。初步数据表明，在产前暴露于四氢大麻酚的成年大鼠中，Drd2基因含有异常的组蛋白H3甲基化标记。本提案的目的是评估产前THC暴露如何通过组蛋白修饰酶改变染色质甲基化，这些酶破坏D2受体的调节和功能，导致敏感性增加，从而在以后的生活中形成成瘾。为了研究组蛋白修饰破坏的机制，将通过qPCR和western blot分析暴露于四氢大麻酚的大鼠的组蛋白修饰酶的mRNA和蛋白水平。鉴定组蛋白修饰酶mRNA水平的纹状体解剖模式将在大麻和来自我们独特的人类胎儿脑库的对照受试者中进行检查。为了确定Drd2还原背后的特定组蛋白修饰酶，将通过在神经细胞系中敲除或过表达来操纵候选蛋白，并研究其对染色质结构和Drd2表达的影响。我们将在产前四氢大麻酚暴露大鼠NAc中进行慢病毒介导的相关组蛋白修饰子表达调节，并评估与海洛因自我给药相关的行为后果，以了解四氢大麻酚诱导的分子变化与表型之间的关系。

DESCRIPTION (provided by applicant): An increasing number of states are legalizing marijuana (Cannabis sativa), yet the neurobiological consequences are largely unknown. During pregnancy, the human fetus is particularly vulnerable to exogenous insults, such as exposure to drugs. To date, cannabis is the most commonly abused illicit drug by pregnant mothers in North America. Epidemiological studies have documented a significant link between repeated early cannabis exposure and an increased risk for subsequent neuropsychiatric abnormalities, including drug addiction and schizoafective disorders. Cannabis is known to target cannabinoid receptors, which are involved in hardwiring the brain during the prenatal period and are expressed on neurons that control dopamine neurotransmission in the nucleus accumbens (NAc). Data from both our unique human fetal brain collection and rat model show a significant decrease in dopaminergic D2 receptor (Drd2) gene expression in the NAc folowing prenatal cannabis/THC exposure. Interestingly, our animal studies demonstrated that this D2 receptor reduction is caused by altered mRNA expression and it persists into adulthood, thus indicating a molecular mechanism, which can sustain a long- term temporal effect that canot be explained by individual genotype. The epigenome controls gene regulation and is influenced by the environment, thus it is a highly relevant biological candidate capable of maintaining aberrant D2 receptor function as a result of developmental drug exposure. A major epigenetic mechanism known to be crucial for neurodevelopment is covalent modifications of nucleosomal histones on chromatin that regulate gene transcription. Preliminary data indicate that the Drd2 gene contains abnormal histone H3 methylation marks in adult rats that were exposed to THC prenatally. The purpose of this proposal is to evaluate how prenatal THC exposure alters chromatin methylation through histone modifying enzymes that disrupt the regulation and function of D2 receptors, leading to increased sensitivity to develop addiction later in life. To investigate the mechanisms underlying histone modification disruption, mRNA and protein levels of histone modifying enzymes will be determined by qPCR and western blot analysis in rats exposed to THC prenatally. The striatal anatomical pattern of the identified histone modifying enzyme mRNA levels will be examined in cannabis and control subjects from our unique human fetal brain bank. To determine the specific histone modifying enzymes behind Drd2 reduction, candidate proteins wil be manipulated via knock-down or overexpression in a neuronal cell line and consequences on the chromatin structure and Drd2 expression will be studied. Lentiviral-mediated modulation of the expression of the relevant histone modifer identified will be conducted in the NAc of rats with prenatal THC exposure and the behavioral consequences evaluated in relation to heroin self- administration to gain insight into the relationship between THC-induced molecular changes and phenotype. PUBLIC HEALTH RELEVANCE: Marijuana is the most commonly abused drug among pregnant women, despite studies that report that fetuses exposed to this drug have an increased susceptibility to psychiatric diseases later in life, including a drug addiction. We have found that marijuana changes chromatin structure in specific brain regions that results in abnormalities in neurotransmitter systems linked to addiction risk. This project serves to evaluate how marijuana causes these irregularities, through the investigation of underlying proteins, to ultimately better understand the mechanism behind addiction vulnerability.