Translatome profiling of nicotine addiction

摘要 吸烟是可预防的首要死因。尼古丁（NIC）决定了 smoking.在大鼠中，NIC添加通常通过静脉内NIC自我给药（SA）建模。我们 (Dr. Vezina）最近表明，NIC致敏，一个过程，出现后，反复暴露于 NIC，可增强NIC等兴奋剂的SA。已知大鼠中的NIC致敏和SA相关 在多个脑区的神经生物学变化，然而，神经生物学的分子基础 NIC敏感性和SA的变化及其与人类NIC滥用的相关性尚未确定。 我们建议利用RNA测序（RNAseq）的深度和广度来识别 通过定量积极参与NIC致敏和SA的转录本的基因表达谱 蛋白质合成（即，translatome）在这些过程中，并探讨其相关性，以增强NIC 用于人类。翻译组分析比常规转录组更好地预测蛋白质丰度 与基于质谱的蛋白质组学测定相比，该方法具有更高的分辨率和动态范围。到 模型NIC致敏和SA，我们将创新地使用两个近交系Fischer-344（F344）的F1后代， 和布朗挪威（BN），因为F1大鼠（1）遗传相同，因此最大限度地减少了“遗传噪音”， 测量的差异表达和（2）在亲本菌株具有不同等位基因的所有基因座处杂合， 从而使用于分析等位基因特异性表达（ASE）的信息杂合变体最大化。我们有 这些F1代大鼠（F344/BN）表现出强烈的NIC致敏性。目标1：我们将描述 通过对与腹侧核糖体结合的RNA转录本进行测序来研究NIC致敏和SA的翻译组谱 被盖区（VTA）和脑桥核（NAc），与NIC成瘾最相关的大脑区域。我们将 进一步鉴定显示致敏相关基因翻译的ASE的遗传变异。目标2：检查 NIC成瘾相关的翻译组概况的相关性增强NIC在人类中的使用，我们将 从吸烟者和非吸烟者中获得诱导多能干细胞（iPSC）和中脑DA神经元， 将DA神经元暴露于NIC，并检查NIC诱导的iPSC神经元表达变化如何与 与在大鼠脑组织中观察到的结果一致。对于与NIC致敏相关的基因，我们将进一步评估 通过在公开场合进行基于路径的关联测试， 吸烟GWAS数据集。鉴定与NIC致敏和SA相关的新基因靶标将 开辟一条新的道路，加深我们对人类滥用NIC的神经生物学的理解，帮助开发更多 有效的治疗措施。

Abstract Smoking is the foremost preventable cause of death. Nicotine (NIC) determines the addictive nature of smoking. In rats, NIC addition has been commonly modeled by intravenous NIC self-administration (SA). We (Dr. Vezina) have recently shown that NIC sensitization, a process that appears after repeated exposure to NIC, can enhance the SA of NIC and other stimulants. NIC sensitization and SA in rats are known to correlate with neurobiological changes in multiple brain regions, however, the molecular basis of the neurobiological changes of NIC sensitization and SA as well as its relevance to human NIC abuse has yet to be determined. We propose to take advantage of the depth and breadth of RNA sequencing (RNAseq) to identify the gene expression profiles of NIC sensitization and SA by quantifying transcripts actively involved in protein synthesis (i.e., translatome) in these processes, and to explore its relevance to enhanced NIC use in humans. Translatome profiling better predicts protein abundance than conventional transcriptome profiling and has higher resolution and dynamic range than mass spectrometry-based proteomic assays. To model NIC sensitization and SA, we will innovatively use F1 progeny of two inbred strains, Fischer-344 (F344) and Brown Norway (BN) since F1 rats are (1) genetically identical, thus minimizing “genetic noise” on measured differential expression and (2) heterozygous at all loci where parental strains have different alleles, thus maximizing the informative heterozygous variants for analyzing allele-specific expression (ASE). We have shown that these F1 rats (F344/BN) exhibit robust NIC sensitization. AIM1: We will characterize the translatome profiles of NIC sensitization and SA by sequencing RNA transcripts bound to ribosomes in ventral tegmental area (VTA) and nucleus accumbens (NAc), the brain regions most relevant to NIC addiction. We will further identify genetic variants showing ASE of sensitization-associated gene translation. AIM2: To examine the relevance of NIC addiction-associated translatome profiles to the enhanced NIC use in humans, we will derive induced pluripotent stem cells (iPSCs) and midbrain DA neurons from both smokers and non-smokers, expose DA neurons to NIC, and examine how the NIC-induced expression changes in iPSC-neurons correlate with those observed in rat brain tissues. For genes correlated with NIC sensitization, we will further evaluate their relevance to human smoking behavior by performing a pathway-based association test in publicly available smoking GWAS datasets. Identifying novel gene targets relevant to NIC sensitization and SA will forge a new path to deepen our understanding of the neurobiology of human NIC abuse, helping develop more effective therapeutic interventions.