Regulatory mechanisms of PDF neuropeptide production in the Drosophila clock neurons

果蝇时钟神经元 PDF 神经肽产生的调节机制

• 批准号: 10114768
• 负责人: JAE H PARK
• 金额: $ 45.82万
• 依托单位: UNIVERSITY OF TENNESSEE KNOXVILLE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-11 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10114768
• 关键词: Adult; Alleles; Alzheimer' s Disease; Animals; Autoimmune Diseases; Base Sequence; Behavior; Behavioral; Binding; Biochemical; Biological Rhythm; Brain; C-terminal; CRISPR interference; CRISPR/Cas technology; Cells; Circadian Rhythms; Cues; Data; Dissection; Drosophila genus; Drosophila melanogaster; Elements; Environment; Event; Feedback; Gene Expression; Gene Expression Profiling; Genes; Genetic; Genetic Transcription; Health; Histologic; Hour; Immunologics; Induced Mutation; Jet Lag Syndrome; Light; Maintenance; Malignant Neoplasms; Maps; Measures; Molecular; Molecular Genetics; Motor Activity; Mutation; Nerve; Nervous system structure; Neuraxis; Neurons; Neuropeptides; Nucleic Acid Regulatory Sequences; Organism; Output; Pacemakers; Pattern; Performance at work; Periodicity; Peripheral; Phenotype; Physiological; Physiology; Pigments; Planet Earth; Point Mutation; Process; Production; Proteins; Regulatory Element; Reporter Genes; Reporting; Role; Rotation; Signal Transduction; Signaling Molecule; Sleep Disorders; Sulfur; Techniques; Temperature; Testing; Time; Tissues; Transcript; Transgenic Organisms; amidation; base; cell type; circadian; environmental change; fly; gain of function; in vivo; insight; loss of function; mutant; neural circuit; novel; receptor; screening; shift work; tool; transcription factor

PROJECT SUMMARY Construction of functional nervous system in the central nervous system (CNS) requires elaborate connectivity of immensely heterogeneous neuronal cell types. One such neural circuit is dedicated to control various biological rhythms with approximately 24-hour periodicity, termed circadian rhythms. Disruption of the rhythms is closely associated with various health-related problems such as sleep disorders, jet-lag, job-performance associated with shifted works, autoimmune disease, cancer, and Alzheimer’s disease. The cellular and molecular bases of the endogenous clock have been well studied particularly in Drosophila. Approximately 150 neurons in the adult Drosophila CNS form a neural circuit regulating the circadian rhythmic behavior and physiology. These clock neurons intimately communicate with each other via secretory factors from these neurons. A major clock messenger molecule is a neuropeptide, pigment dispersing factor (PDF), which is produced by a group of major clock neurons, s- LNvs. Despite these studies, how Pdf gene expression is regulated and how the PDF precursor molecules are processed to secrete functionally mature PDF are not well understood. These regulatory mechanisms are certainly important for timely production of PDF to regulate circadian rhythms. From our studies on the transcriptional mechanism of Pdf, we have recently reported a 35-bp Pdf regulatory element (PRE) that is essential for the Pdf expression. To gain further insight into the mechanisms underlying PDF production in the clock neurons, we propose the following aims: (1) Functional dissection of PRE in vivo. Using CRISPR/Cas9 and CRISPR interference (CRISPRi) tools, we will demonstrate the endogenous role of PRE in vivo and determine s-LNv-specific element within PRE. (2) Screening Pdf- regulating transcription factors. We will employ both gain-of-function and loss-of-function strategies to identify potential candidates that bind to PRE and thus regulate Pdf expression. (3) Clock-controlled processing of PDF precursor. Functionally mature PDF is part of a large precursor, which is predicted to be processed via proteolytic cleavage and modified by C-terminal amidation. Our previous data suggest that such post-translational events might be clock-controlled to release PDF. We will develop a novel immunological as well as various genetic and transgenic tools to study the mechanisms of PDF maturational process. (4) Characterization of a new Clock (Clk) mutant allele. Clk gene is a central clock factor that also regulates Pdf expression indirectly. Recently we have found a new arrhythmic Clk mutant allele, which we call tentatively no-time (Clknot). Molecular characterization of this new mutant allele will shed more light on the functional aspects of CLK protein.

项目摘要 中枢神经系统（CNS）功能神经系统的构建需要精细的 非常异质的神经元细胞类型的连接。一个这样的神经回路致力于 以大约24小时的周期控制各种生物节律，称为昼夜节律。 节律的破坏与各种健康相关的问题密切相关，例如睡眠 疾病，时差，与轮班工作相关的工作表现，自身免疫性疾病，癌症， 老年痴呆症内源性生物钟的细胞和分子基础已经得到了很好的研究 尤其是果蝇。成年果蝇中枢神经系统中大约有150个神经元组成一个神经回路 调节昼夜节律行为和生理。这些生物钟神经元紧密地 通过这些神经元的分泌因子相互作用。一个主要的时钟信使分子是 神经肽，色素分散因子（PDF），这是由一组主要的时钟神经元，s- LNV。尽管有这些研究，但Pdf基因表达是如何调节的，PDF前体是如何被调节的。 分子被加工以分泌功能成熟的PDF还没有被很好地理解。这些监管 这些机制对于及时产生PDF以调节昼夜节律当然是重要的。从我们 为了研究Pdf的转录机制，我们最近报道了一个35 bp的Pdf调控基因， 元素（PRE），它是Pdf表达式所必需的。为了更深入地了解 在时钟神经元的PDF产生的基础上，我们提出了以下目标：（1）功能解剖 体内的PRE。使用CRISPR/Cas9和CRISPR干扰（CRISPRi）工具，我们将展示 PRE在体内的内源性作用和确定PRE内的s-LNv特异性元件。(2)筛选PDF- 调节转录因子。我们将采用功能获得和功能丧失策略， 鉴定与PRE结合从而调节Pdf表达的潜在候选物。(3)钟控 PDF前体的处理。功能成熟的PDF是一个大的前体的一部分，预计将 通过蛋白水解切割进行加工并通过C-末端酰胺化进行修饰。我们之前的数据显示 这种翻译后事件可能是时钟控制的，以释放PDF。我们要写一部小说 免疫学以及各种遗传和转基因工具来研究PDF的机制 成熟过程(4)一种新的Clock（Clk）突变等位基因的表征。Clk基因是一个中心时钟 该因子也间接调节Pdf表达。最近，我们发现了一个新的细胞Clk突变体， 我们暂时称之为无时间（Clknot）。这种新的突变等位基因的分子特征将 对CLK蛋白的功能方面有更多的了解。