GEF activity in circadian pacemaker neurons

描述（由申请人提供）：研究昼夜节律（~24小时）节律提供了一个极好的机会，可以在分子，细胞和电路水平上了解关键的大脑功能，并可能确定治疗睡眠障碍和时差的新靶点。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，与遗传性人类睡眠障碍有关。时钟基因在中央脑起搏器神经元中起作用，控制整个动物的行为节律。起搏器神经元的内源性分子和神经节律为研究基因表达如何控制神经元信号的日常变化提供了一个独特的模型，至少在果蝇中，包括结构可塑性的节律。为了确定新的昼夜节律调节因子，我们从一组纯化的主起搏器神经元（果蝇LNvs）中生成了全基因组表达谱。我们确定了一组10个基因，这些基因以时钟依赖的方式以每日节律表达，并且在LNvs中比在其他分化神经元中表达得更高。在这10个基因中，有4个编码了先前确定的核心时钟基因，如周期。本文拟对另外6个基因之一的CG33275进行研究。CG33275是一个尚未研究的基因，可能编码激活Rho家族GTPase的鸟嘌呤核苷酸交换因子（guine nucleotide Exchange Factor， GEF）。CG33725是人类紫癜蛋白的果蝇同源基因，与脊髓小脑性共济失调的遗传形式有关，但尚未在分子水平上进行研究。我们将CG33275称为dPuratrophin (dPura)，并认为对该基因在果蝇中的基础研究有助于解释人类Puratrophin的疾病相关性。节律性dPura表达可能会对Rho家族GTPase的活性施加昼夜节律，而Rho家族GTPase也尚未与昼夜节律有关。在这里，我们首先利用遗传学和生物化学来确定dPura是否确实是一种GEF。利用遗传学，我们将测试6个果蝇Rho GTPase家族成员中的哪一个与时钟神经元中的dPura基因相互作用以调节昼夜节律行为。我们将用直接测量dPura GEF活性的体外生化实验来补充这些体内实验。我们的第二个目标是通过表征我们已经确定的强烈改变昼夜行为节律的dPura突变体来确定dPura在LNvs中的作用。具体来说，我们将询问dPura突变体是否显示LNvs中昼夜节律基因表达、结构可塑性和/或细胞内运输的改变，这可能是行为缺陷的基础。由于gef经常被细胞外信号激活，dPura可以帮助起搏器神经元整合内部时钟时间（通过节律表达）与外部信号。鉴于小鼠的Puratrophin在大脑中也显示出昼夜节律表达，我们的研究应该能够深入了解苍蝇和哺乳动物的昼夜节律，并有助于了解Puratrophin的一般功能。

DESCRIPTION (provided by applicant): Studying circadian (~24hr) rhythms offers an excellent opportunity to understand a key brain function at molecular, cellular and circuit levels as well as possibly identifying novel targets for therapies for sleep disorders and jetlag. Studies in Drosophila identified the first circadian clock gene, which is conserved in humans and linked to an inherited human sleep disorder. Clock genes function in central brain pacemaker neurons to control whole animal behavioral rhythms. The endogenous molecular and neural rhythms of pacemaker neurons provide a unique model to study how gene expression controls daily changes in neuronal signaling which, at least in Drosophila, includes rhythms in structural plasticity. To identify novel regulators of circadian rhythms, we generated whole genome expression profiles from a group of purified master pacemaker neurons, the Drosophila LNvs. We identified a set of 10 genes that are expressed with a daily rhythm in a clock-dependent manner and which are much more highly expressed in LNvs than in other differentiated neurons. Of these 10 genes, four encode previously identified core clock genes such as period. Here, we propose to study CG33275, one of the other 6 genes. CG33275 is an unstudied gene which likely encodes a Guanine nucleotide Exchange Factor (GEF) that activates a Rho family GTPase. CG33725 is the Drosophila ortholog of human Puratrophin, which has been linked with a hereditary form of spinocerebellar ataxia, but is unstudied at the molecular level. We refer to CG33275 as dPuratrophin (dPura) and believe that basic studies of this gene in Drosophila could help explain the disease-association of human Puratrophin. Rhythmic dPura expression could impose circadian rhythms on the activity of a Rho family GTPase, which have also not yet been implicated in circadian rhythms. Here, we first aim to determine if dPura is indeed a GEF using genetics and biochemistry. Using genetics, we will test which of the 6 Drosophila Rho GTPase family members genetically interact with dPura in clock neurons to regulate circadian behavior. We will complement these in vivo experiments with in vitro biochemical experiments that directly measure dPura GEF activity. Our second aim is to identify the role of dPura in LNvs by characterizing dPura mutants we have identified that strongly alter circadian behavioral rhythms. Specifically, we will ask if dPura mutants show altered circadian gene expression, structural plasticity and/or intracellular trafficking in LNvs that could underlie the behavioral defects. Since GEFs are often activated by extracellular signals, dPura could help pacemaker neurons integrate internal clock time (via rhythmic expression) with external signals. Given that mouse Puratrophin also shows circadian expression in the brain, our studies should give insight into both fly and mammalian circadian rhythms as well as helping understand Puratrophin function in general. PUBLIC HEALTH RELEVANCE: Circadian pacemaker neurons are an excellent model to study the genetic contribution to changes in neuronal activity and plasticity. We have identified a novel regulator in pacemaker neurons - dPura, the fly ortholog of a gene associated with spinocerebellar ataxia in humans. We propose to identify dPura's molecular target in pacemaker neurons (likely conserved in humans) and how dPura regulates pacemaker neuron function.