Regulation for Pacemaker Neurons

描述（由申请人提供）：研究昼夜节律（~24小时）提供了一个巨大的机会来了解分子，细胞和电路水平的基本大脑功能，以及新疗法的机会。对果蝇的研究发现了第一个生物钟基因，该基因在人类中是保守的，并在家族性睡眠障碍中发生突变。生物钟基因在大脑的起搏器神经元中起作用，驱动行为节律。尽管我们对这些分子钟有了相对较好的了解，但分子钟如何调节有节奏的神经元活动以及有节奏的输出如何反馈给分子钟仍是未知的。为了确定起搏器神经元的潜在输出，我们对一组同质的主起搏器神经元（果蝇LNvs）进行了基因表达谱研究。LNvs中节律性表达的一个基因编码一个内向整流K+通道（Ir）。在Aim 1中，我们验证了核心时钟转录因子直接调节Ir表达以调节LNv兴奋性的观点。LNvs中Ir表达的改变改变了昼夜节律的周期长度。因此LNvs的电活动反馈来调节它们的分子钟。为了测试这种机制是否与转录有关，我们测量了组成型超极化或超兴奋的LNvs中的基因表达谱。我们发现昼夜节律基因的表达发生了戏剧性的重组，这样，在黎明，当LNvs通常最兴奋时，它们的超极化使它们的昼夜节律基因表达类似于黄昏，而它们通常是不活跃的。相反，黄昏时高度兴奋的LNvs使其昼夜节律基因表达与黎明相似。因此，LNv神经元活动可以对昼夜节律基因表达施加时间，这表明我们对昼夜节律分子钟的理解有了重大修订。在目标2中，我们建议确定将神经元活动与基因表达的昼夜节律联系起来的转录因子和信号通路。一组基因通过改变LNv的兴奋性而被错误调控，编码翻译起始调控因子。这是惊人的，因为三种蛋白质翻译调节因子在LNvs中高度表达，这些因子的突变改变了昼夜节律和/或光重置时钟。因此，翻译起始是一个以前未被认识到的分子钟调控步骤。在Aim 3中，我们建议测试这些翻译调节因子是否影响LNvs中特定时钟mrna的翻译和/或全局翻译。我们还将测试这些调节因子是否对光和神经元活动等信号作出反应，从而对LNvs中的翻译进行剧烈调节。我们相信LNvs的内源性神经和分子节律，结合我们开发的来自完整神经系统的LNvs全基因组分析技术，使LNvs成为研究基因表达与神经元兴奋性和可塑性之间动态双向关系的独特模型。

DESCRIPTION (provided by applicant): Studying circadian (~24hr) rhythms offers a tremendous opportunity to understand a fundamental brain function at molecular, cellular and circuit levels as well as opportunities for novel therapies. Studies in Drosophila identified the first circadian clock gene, which is conserved in humans and is mutated in a familial sleep disorder. Clock genes function within pacemaker neurons in the brain to drive behavioral rhythms. Although we have a relatively good understanding of these molecular clocks, it is unknown how molecular clocks regulate rhythmic neuronal activity and how rhythmic outputs feed back to the molecular clock. To identify potential outputs of pacemaker neurons, we conducted gene expression profiling studies from a group of homogeneous master pacemaker neurons, the Drosophila LNvs. One gene rhythmically expressed in LNvs encodes an Inward rectifier K+ channel (Ir). In Aim 1, we test the idea that Ir expression is directly regulated by the core clock transcription factors to regulate LNv excitability. Altered Ir expression in LNvs changes the period length of circadian rhythms. Thus the electrical activity of LNvs feeds back to regulate their molecular clocks. To test if the mechanism for this is transcriptional, we measured gene expression profiles in LNvs that were either constitutively hyperpolarized or hyperexcited. We found dramatic re-organization of circadian gene expression such that hyperpolarizing LNvs at dawn when they are normally most excitable makes their circadian gene expression resemble dusk, when they are normally inactive. Conversely, hyperexciting LNvs at dusk makes their circadian gene expression resemble dawn. Thus LNv neuronal activity can impose time-of-day to circadian gene expression and this suggests a major revision to our understanding of circadian molecular clocks. In Aim 2, we propose to identify the transcription factor(s) and signaling pathway(s) that connect neuronal activity to circadian rhythms in gene expression. One group of genes mis-regulated by altering LNv excitability encode translation initiation regulators. This was striking because three protein translation regulators are highly expressed in LNvs and mutants in these factors alter circadian rhythms and/or clock resetting by light. Thus translation initiation is a previously unappreciated regulatory step in the molecular clock. In Aim 3, we propose to test if these translation regulators affect translation of specific clock mRNAs and/or global translation in LNvs. We will also test if these regulators respond to signals such as light and neuronal activity to acutely regulate translation in LNvs. We believe that the endogenous neural and molecular rhythms of LNvs, combined with techniques we developed for whole-genome profiling of LNvs from an intact nervous system, make LNvs a unique model to study the dynamic bi-directional relationships between gene expression and neuronal excitability and plasticity. PUBLIC HEALTH RELEVANCE: Disrupted circadian (~24hr) rhythms are associated with a wide range of human symptoms from sleep and mood disorders to increased incidences of cancer and obesity. These rhythms are controlled by intracellular clocks whose understanding at the molecular level may open up novel therapies. Here we seek to understand the molecules that regulate molecular clock speed via electrical activity and protein translation.