Translatome profiling of nicotine addiction

摘要 吸烟是最重要的可预防的死亡原因。尼古丁(NIC)决定了尼古丁的成瘾性质 抽烟。在大鼠中，NIC添加通常通过静脉注射NIC自我给药(SA)来模拟。我们 (Vezina博士)最近发现NIC敏化，这是一种在反复接触 NIC，可增强NIC等兴奋剂的SA。已知大鼠的NIC敏化和SA相关 然而，随着神经生物学在多个脑区的变化，神经生物学的分子基础 NIC敏化和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 NIC致敏和SA基因表达谱研究 这些过程中的蛋白质合成(即翻译组)，并探讨其与增强型NIC的相关性 在人类身上使用。翻译组谱分析比传统转录组更好地预测蛋白质丰度 与基于质谱学的蛋白质组分析相比，该方法具有更高的分辨率和动态范围。至 模型NIC敏化和SA，我们将创新性地使用两个近交系Fischer-344(F344)的F1后代 和棕色挪威(BN)，因为F1大鼠在(1)基因上是相同的，因此最大限度地减少了 在亲本菌株具有不同等位基因的所有基因座上测量差异表达和(2)杂合子， 从而最大化用于分析等位基因特异性表达(ASE)的信息丰富的杂合变体。我们有 结果表明，这些F1大鼠(F344/BN)表现出较强的NIC敏感性。AIM1：我们将描述 腹侧核糖体结合RNA转录本测序研究NIC致敏和SA的翻译组特征 被盖区(VTA)和伏隔核(NAC)，这是大脑中与NIC成瘾最相关的区域。我们会 进一步鉴定表现致敏相关基因翻译酶活性的遗传变异。AIM2：检查 与NIC成瘾相关的翻译组配置文件与人类NIC使用的增强相关，我们将 从吸烟者和非吸烟者身上获得诱导多能干细胞(IPSCs)和中脑DA神经元， 将DA神经元暴露于NIC，并研究NIC诱导的IPSC神经元表达变化是如何相互关联的 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 通过在公共场合进行基于途径的关联测试，它们与人类吸烟行为的相关性 可用的吸烟GWAs数据集。寻找与NIC增敏和SA相关的新基因靶点 打造一条新的途径来加深我们对人类NIC滥用的神经生物学的理解，帮助开发更多 有效的治疗干预。

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.