Exploiting zebrafish genetics to identify genes affecting addiction-related phenotypes.

利用斑马鱼遗传学鉴定影响成瘾相关表型的基因 据估计，仅在美国，每年因药物滥用而造成的可预防健康损失就高达1660亿美元。 医疗费用，其中烟草（1300亿美元）和酒精（250亿美元）滥用占压倒性的比例 大部分的费用。世界卫生组织宣布烟草相关疾病为全球性疾病， 这一流行病如果不加遏制，预计到2030年将在全世界每年造成约800万人死亡。 有一个至关重要的需要增加了解的遗传和细胞生物学因素的影响 易成瘾性，包括吸烟，以帮助识别有风险的个人，并为治疗提供信息 战略布局由于确定人类遗传风险的方法很困难，因此可以对遗传风险进行研究。 通过使用动物模型来促进。斑马鱼是一种成熟的发育和行为 对常见药物滥用和行为表现出保守反应的遗传模式物种 模拟出易成瘾的核心表型我们将识别影响核心的基因， 通过筛选乙基亚硝基脲（ENU）诱变的品系， 对尼古丁奖赏的敏感性和冲动性。我们检验了一个假设， 慢性尼古丁暴露后表达的持续适应性变化也是成瘾的原因之一。 脆弱基因 我们的具体目标是： 1)分离影响a）尼古丁奖赏和B）冲动性的25个遗传位点。 i）我们将在成年ENU诱变的斑马鱼中使用行为测定来鉴定家族 显示出尼古丁奖赏和冲动控制的改变我们已经开发了一个可扩展的，完全 自动化行为分析系统用于此目的。我们将使用微阵列分析 或RNAseq数据，以鉴定在以下情况下表现出持续表达变化的基因： 慢性尼古丁暴露作为影响尼古丁成瘾基因的候选者 易损性.我们将预先加载带有这些突变的家庭的屏幕， 候选基因 ii）我们将使用外显子组测序来定位至少10个家族中的突变。使用 ENU诱变的细胞系有助于鉴定致病突变（通过产生突变）。 强表型和/或作为标记的ENU突变）。 2)确定所观察到的行为表型背后的细胞生物学过程。 i）我们将使用额外的（预测的功能性）ENU突变或靶向敲除的基因。 鉴定基因以确认基因型-表型关联。 ii）我们将进行RNA测序，以深入了解对基因的可能影响。 表达和细胞通路受到影响。 iii）我们将进行发展分析，以检验行为影响的假设， 是通过对神经元模式或神经回路形成的影响来介导的， 发展时间点。 本研究的预期结果是确定影响核心的新的遗传因素。 行为预测药物成瘾的脆弱性，并增加对细胞的理解， 进程受到影响。这些发现将使未来的人类遗传变异研究在这些确定的 基因，有可能开发出更个性化的疗法。这项工作的结果将有一个 对烟草依赖和成瘾的个体化治疗工作产生积极影响。

Exploiting zebrafish genetics to identify genes affecting addiction-related phenotypes The cost of substance abuse in the US alone is estimated at $166 billion annually in preventable health care costs, with tobacco ($130 billion) and alcohol ($25 billion) abuse contributing to the overwhelming majority of this expense. The World Health Organization declared tobacco-related disease a global epidemic, predicted to cause an estimated 8 million annual deaths worldwide by 2030, if unabated. There is a vital need for increased understanding of the genetic and cell biological factors influencing vulnerability to addiction including smoking to help identify individuals at risk and to inform treatment strategies. As approaches to identify genetic risk are difficult in humans, research on genetic risk can be facilitated by the use of animal models. Zebrafish are an established developmental and behavioral genetic model species that show conserved responses to common drugs of abuse as well as behaviors that model core phenotypes predictive of vulnerability to addiction. We shall identify genes affecting core phenotypes associated with addiction by screening lines of ethyl nitrosurea (ENU)-mutagenised zebrafish for sensitivity to nicotine reward and impulsivity. We test the hypothesis that genes that show persistent adaptive changes in expression following chronic nicotine exposure are also addiction vulnerability genes. Our specific aims are: 1) To isolate 25 heritable loci affecting a) nicotine reward and b) impulsivity. i) We will use behavioral assays in adult ENU-mutagenized zebrafish to identify families showing altered nicotine reward and impulse control. We have developed a scalable, fully automated behavioral assay system for this purpose. We will use analysis of microarray or RNAseq data to identify genes that show persistent changes in expression following chronic nicotine exposure as candidates for genes affecting nicotine addiction vulnerability. We will pre-load the screen with families carrying mutations in these candidate genes. ii) We shall use exome sequencing to map the mutations in at least 10 families. The use of ENU- mutagenized lines facilitates identification of the causal mutations (by generation of strong phenotypes and/or by the ENU mutations acting as markers). 2) To identify the cell biological processes underlying the observed behavioral phenotypes. i) We shall use additional (predicted functional) ENU mutations or targeted knockouts of the identified genes to confirm genotype-phenotype associations. ii) We shall perform RNA sequencing to gain insight into possible effects on gene expression and cellular pathways affected. iii) We shall perform developmental analyses to test the hypothesis that behavioral effects are mediated by effects on neuronal patterning or neural circuit formation at developmental timepoints. The expected outcomes from this study are identification of novel genetic factors influencing core behaviors predictive of vulnerability to drug addiction and increased understanding of the cellular processes affected. These findings will enable future human genetic variation studies in these identified genes, with the potential to develop more personalized therapies. Results from this work will have a positive impact on individualized treatment efforts for tobacco dependence and addiction as a whole.