GEF activity in circadian pacemaker neurons

昼夜节律起搏神经元中的 GEF 活性

• 批准号: 8320129
• 负责人: JUSTIN BLAU
• 金额: $ 7.31万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8320129
• 关键词: Action Potentials; Address; Adult; Affect; Animal Behavior; Animals; Behavior; Behavioral; Biochemical; Biochemistry; Biological Assay; Biological Clocks; Brain; Brain region; Cells; Circadian Rhythms; Complement; Complex; Darkness; Data; Data Set; Defect; Disease Association; Drosophila genus; Family; Family member; Freezing; Frequencies; Gene Expression; Gene Proteins; Genes; Genetic; Genome; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Human; In Vitro; Inherited; Jet Lag Syndrome; Lead; Link; Mammals; Measures; Membrane Potentials; Methods; Mitochondria; Modeling; Molecular; Molecular Profiling; Molecular Target; Mus; Mutation; Neuronal Plasticity; Neurons; Orthologous Gene; Pacemakers; Pattern; Periodicity; Phase; Phenotype; Physiology; Population; Process; RNA Interference; Reading; Research; Rest; Role; Signal Transduction; Sleep Disorders; Sleep Disorders Therapy; Spinocerebellar Ataxias; Testing; Time; Transgenes; base; circadian behavioral rhythms; circadian pacemaker; extracellular; fly; gene function; in vivo; insight; meetings; mutant; novel; programs; relating to nervous system; research study; rho; rho GTP-Binding Proteins; trafficking

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.

描述（由申请人提供）：研究昼夜（~ 24小时）节律提供了一个极好的机会，可以在分子、细胞和回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。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，并与遗传性人类睡眠障碍有关。生物钟基因在中枢脑起搏神经元中起作用，控制整个动物的行为节奏。起搏神经元的内源性分子和神经节律提供了一个独特的模型来研究基因表达如何控制神经元信号传导的日常变化，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器，我们从一组纯化的主起搏神经元（果蝇LNvs）中产生了全基因组表达谱。我们确定了一组10个基因，它们以时钟依赖的方式以每日节律表达，并且在LNvs中的表达比在其他分化的神经元中高得多。在这10个基因中，有4个编码先前确定的核心时钟基因，如周期。在这里，我们建议研究其他6个基因之一的CG 33275。CG 33275是一个未经研究的基因，它可能编码一种激活Rho家族GT3的鸟嘌呤核苷酸交换因子（GEF）。CG 33725是人类Puratrophin的果蝇直系同源物，其与脊髓小脑共济失调的遗传形式有关，但未在分子水平上进行研究。我们将CG 33275称为dPuratrophin（dPura），并认为对果蝇中该基因的基础研究有助于解释人类Puratrophin的疾病相关性。 节律性dPura表达可以对Rho家族GTdR的活性施加昼夜节律，其也尚未涉及昼夜节律。在这里，我们首先要确定dPura是否确实是一个使用遗传学和生物化学的GEF。利用遗传学，我们将测试6个果蝇Rho GTdR家族成员中的哪一个在生物钟神经元中与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确定dPura在LNvs中的作用，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。具体来说，我们将询问dPura突变体是否显示改变的昼夜节律基因表达，结构可塑性和/或LNvs的细胞内运输，这可能是行为缺陷的基础。由于GEF通常由细胞外信号激活，dPura可以帮助起搏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Puratrophin也显示出大脑中的昼夜节律表达，我们的研究应该深入了解苍蝇和哺乳动物的昼夜节律，以及帮助了解Puratrophin的一般功能。