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

利用斑马鱼遗传学识别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达1660亿美元 护理成本，其中烟草(1300亿美元)和酒精(250亿美元)滥用造成了压倒性的 这笔费用的大部分。世界卫生组织宣布与烟草有关的疾病是全球 预计到2030年，如果疫情不减，每年将导致全球约800万人死亡。 有必要增加对影响基因和细胞生物学因素的了解 易上瘾，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。由于识别人类遗传风险的方法很困难，对遗传风险的研究可能会 通过使用动物模型来促进这一过程。斑马鱼是一种成熟的发育和行为 对常见滥用药物和行为表现出保守反应的遗传模式物种 该模型预测了易上瘾的核心表型。我们将确定影响核心的基因 乙基亚硝脲诱变品系筛选与成瘾相关的表型 斑马鱼对尼古丁奖励和冲动的敏感性。我们测试了这样一种假设，即基因表明 慢性尼古丁暴露后表达的持续适应性变化也是成瘾 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)冲动的25个可遗传基因座。 I)我们将使用ENU诱变的成年斑马鱼的行为分析来鉴定家族 表现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 用于此目的的自动行为分析系统。我们将使用基因芯片的分析 或RNAseq数据来识别在以下情况下显示持续表达变化的基因 慢性尼古丁暴露是影响尼古丁成瘾的候选基因 脆弱性。我们将在屏幕上预先加载携带这些基因突变的家庭 候选基因。 Ii)我们将使用外显子组测序来绘制至少10个家系的突变图谱。对.的使用 Enu诱变的品系有助于鉴定原因突变(通过生成 强表型和/或通过ENU突变作为标记)。 2)确定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)ENU突变或靶向敲除 确定基因以确认基因型与表型的关联。 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。 三)我们将进行发展分析，以检验行为影响的假设 是通过对神经元构型或神经回路形成的影响而介导的 发育时间点。 这项研究的预期结果是确定影响核心的新的遗传因素 预示药物成瘾易感性的行为和对细胞的更多了解 受影响的进程。这些发现将使未来对这些已识别的人类基因变异的研究成为可能 基因，有可能开发更个性化的治疗方法。这项工作的结果将会有一个 对整个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.