Regulation for Pacemaker Neurons

描述(由申请者提供)：研究昼夜节律(~24小时)提供了在分子、细胞和电路水平上了解基本大脑功能的巨大机会，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，这种基因在人类中是保守的，在一种家族性睡眠障碍中发生了突变。时钟基因在大脑中的起搏器神经元中发挥作用，以驱动行为节奏。虽然我们对这些分子钟有了相对较好的了解，但尚不清楚分子钟如何调节节律神经元的活动，以及节律输出如何反馈到分子钟。为了确定起搏神经元的潜在输出，我们对一组同源的起搏主神经元--果蝇LNv进行了基因表达谱研究。在LNV中节律性表达的一个基因编码内向整流钾通道(Ir)。在目标1中，我们验证了IR表达直接由核心时钟转录因子调节LNV兴奋性的想法。LNV中ir表达的改变改变了昼夜节律的周期长度。因此，LNV的电活动反馈调节它们的分子时钟。为了测试这一机制是否与转录有关，我们测量了结构性超极化或过度兴奋的LNV的基因表达谱。我们发现昼夜节律基因表达的戏剧性重组，使得在黎明通常最兴奋的时候超极化的LNV使它们的昼夜基因表达类似于黄昏，而它们通常是不活跃的。相反，在黄昏时过度兴奋的LNV使它们的昼夜基因表达类似黎明。因此，LNV神经元的活动可以施加昼夜节律基因表达的时间，这表明我们对昼夜分子时钟的理解有了重大修订。在目标2中，我们建议确定在基因表达中将神经元活动与昼夜节律联系起来的转录因子(S)和信号通路(S)。一组基因通过改变LNV的兴奋性而被错误调节，编码翻译起始调节因子。这是惊人的，因为三种蛋白质翻译调节因子在LNV中高度表达，并且这些因子中的突变体通过光改变昼夜节律和/或时钟重置。因此，翻译的启动是分子时钟中以前不为人知的调节步骤。在目标3中，我们建议测试这些翻译调节因子是否影响特定时钟mRNAs的翻译和/或LNV中的全局翻译。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做出反应，以敏锐地调节LNV的翻译。我们认为，LNV的内源性神经和分子节律，结合我们开发的完整神经系统LNV的全基因组图谱技术，使LNV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.