Regulatory mechanisms of PDF neuropeptide production in the Drosophila clock neurons

项目总结 中枢神经系统(CNS)功能神经系统的构建需要详细阐述 极其异质的神经细胞类型的连接性。一种这样的神经电路专门用于 以大约24小时的周期控制各种生物节律，称为昼夜节律。 节律紊乱与睡眠等各种与健康相关的问题密切相关 失调症、时差反应、与倒班相关的工作表现、自身免疫性疾病、癌症和 阿尔茨海默氏症。内源性时钟的细胞和分子基础已经得到了很好的研究 尤其是在果蝇身上。成年果蝇中枢神经系统中约150个神经元形成神经回路 调节昼夜节律的行为和生理。这些时钟神经元密切地交流 通过这些神经元的分泌因子相互联系。一个主要的时钟信使分子是一个 神经肽，色素分散因子，由一组主要的时钟神经元产生，S- LNV。尽管有这些研究，PDF基因的表达是如何调节的，PDF前体是如何 对分子进行处理以分泌功能成熟的PDF还没有被很好地理解。这些监管规定 机制对于及时产生PDF以调节昼夜节律当然是重要的。从我们的 关于PDF转录机制的研究，我们最近报道了一种35bp的PDF调控 元素(PRE)，这是PDF表达式所必需的。以进一步深入了解这些机制 在时钟神经元产生PDF的基础上，我们提出了以下目标：(1)功能解剖 体内的前病毒。使用CRISPR/CAS9和CRISPR干扰(CRISPRi)工具，我们将演示 Pre在体内的内源性作用，并确定Pre内S-LNV特异性元件。(2)筛选PDF- 调节转录因子。我们将同时使用功能收益和功能损失策略来 识别与Pre结合的潜在候选者，从而调控PDF表达。(3)时钟控制 PDF前驱体的加工。功能成熟的PDF是一个大型前身的一部分，据预测 通过蛋白水解性裂解和C-端酰胺化进行修饰。我们之前的数据表明 这样的翻译后事件可能是时钟控制的，以发布PDF。我们将写一部小说 免疫学以及各种遗传和转基因工具研究PDF的机制 成熟的过程。(4)一个新的时钟(CLK)突变等位基因的特征。CLK基因是一个中枢时钟 也间接调节PDF表达的因素。最近我们发现了一种新的心律失常clk突变体。 等位基因，我们暂时称之为无时间(克努特)。这一新突变等位基因的分子特征将 进一步阐明了CLK蛋白的功能方面。

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.