Regulation for Pacemaker Neurons

描述（由申请人提供）：研究昼夜（~ 24小时）节律提供了一个巨大的机会，以了解在分子，细胞和电路水平的基本脑功能，以及新的治疗方法的机会。在果蝇中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，在家族性睡眠障碍中发生突变。生物钟基因在大脑中的起搏神经元中起作用，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有节律的神经元活动，以及有节律的输出如何反馈到分子钟。 为了确定起搏神经元的潜在输出，我们进行了基因表达谱研究，从一组同质主起搏神经元，果蝇LNvs。在LNvs中节律性表达的一个基因编码内向整流钾离子通道（Ir）。在目标1中，我们测试的想法，Ir的表达是直接调节的核心时钟转录因子，以调节LNv的兴奋性。 LNvs中Ir表达的改变改变了昼夜节律的周期长度。因此，LNvs的电活动反馈调节其分子钟。为了测试这种机制是否是转录的，我们测量了组成性超极化或超兴奋的LNvs中的基因表达谱。我们发现了昼夜节律基因表达的戏剧性重组，使得在黎明时通常最兴奋的超极化LNvs使它们的昼夜节律基因表达类似于黄昏时，它们通常不活动。相反，黄昏时过度兴奋的LNvs使其昼夜节律基因表达类似于黎明。因此，LNv神经元的活动可以施加时间的昼夜节律基因的表达，这表明了一个重大的修订，我们的理解昼夜节律的分子钟。在目标2中，我们提出了确定的转录因子（S）和信号通路（S）的神经元活动的昼夜节律基因表达。 通过改变LNv兴奋性而错误调节的一组基因编码翻译起始调节子。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，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，翻译起始是一个以前不受重视的调节步骤的分子时钟。在目标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器是否响应信号，如光和神经元活动，以急性调节LNvs的翻译。 我们认为，内源性神经和分子节奏的LNvs，结合我们开发的技术，从一个完整的神经系统的LNvs的全基因组分析，使LNvs一个独特的模型来研究基因表达和神经元的兴奋性和可塑性之间的动态双向关系。

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.