Roles of a novel immunoglobulin-domain protein in sleep and circadian behavior

 描述(申请人提供)：在哺乳动物和昆虫中，多肽和蛋白质激素的细胞间信号对调节睡眠和昼夜行为的回路功能至关重要。在这一应用中，我们提出了实验来验证这样的假设，即一种新的免疫球蛋白(Ig)域蛋白的神经元分泌和作用是果蝇昼夜节律和睡眠回路正常发挥功能所必需的。免疫球蛋白超家族分子是神经发育、突触形成、可塑性和损伤的重要媒介(1；2)，尽管对这些蛋白质的成体功能知之甚少。在最近的表达谱研究中，我们鉴定了许多在成年大脑中表达的果蝇Ig蛋白编码基因。其中包括CG141441(NKT)基因在内的16个基因具有高度的成人神经系统表达，并编码被预测为分泌型分子的Ig蛋白。(FlyAtlas；(3))。在未发表的研究中，我们已经表明，NKT的RNAi产生的泛神经元缺陷的果蝇表现出夜间睡眠减少和昼夜节律延长。选择性地敲除时钟神经元中的NKT会导致昼夜节律延长，而不会影响睡眠。这些结果表明，NKT在不同类型的神经细胞中是调节睡眠和昼夜行为所必需的。有趣的是，基因与一个时期的交互作用(PER) 突变提示NKT作用于起搏神经元，并通过分子时钟调节昼夜节律。由于NKT被预测为一种分泌的小Ig蛋白，我们假设它在昼夜节律中起着类似于PDF神经肽的信号功能。在这 R21的应用，我们建议研究NKT在成人节律行为和睡眠调节中的作用。目标1将使用条件表达方法来验证NKT蛋白在成人大脑中对行为调节的生理需求。此外，它还 将描述神经元在睡眠和昼夜行为中对蛋白质的需求。Aim 2将研究基因与时钟和睡眠改变突变的相互作用，以更好地了解NKT在相关神经元回路中的机械功能。这一目的还将询问NKT是否由神经元分泌，并检验Ig蛋白是分子振荡器功能或时钟神经元种群同步所必需的假设。与NKT同源的蛋白具有单一的免疫球蛋白结构域，存在于许多物种中，包括线虫、果蝇和人类(例如，分泌的小NeuRegin-2亚型)。因此，这项研究可能会对分化大脑中起作用的信号机制提供新的见解。

 DESCRIPTION (provided by applicant): In mammals and insects, intercellular signaling by peptides and protein hormones is critical for the functions of circuits regulating sleep and circadian behavior. In this application, we propose experiments to test the hypothesis that neuronal secretion and action of a novel immunoglobulin (Ig)-domain protein is required for proper function of the Drosophila circadian and sleep circuits. Ig superfamily molecules are important mediators of neural development, synapse formation, plasticity and injury (1;2), although less is known about the adult functions for these proteins. In recent expression profiling studies, we identified numerous Drosophila Ig protein-encoding genes that are expressed in the adult brain. Sixteen of them, including the CG141441 (Nkt) gene, have high adult nervous system expression and encode Ig proteins that are predicted to be secreted molecules. (FlyAtlas; (3)). In unpublished studies, we have shown that flies with RNAi-generated pan-neuronal deficits for Nkt exhibit reduced nighttime sleep and lengthened circadian period. Selective knockdown of Nkt in clock neurons causes a lengthened circadian period with no effect on sleep. These results suggest that Nkt is required in different neuronal cell types for th regulation of sleep and circadian behavior. Interestingly, genetic interaction with a period (pers) mutation suggests that NKT acts on pacemaker neurons and through the molecular clock to regulate circadian period. As NKT is predicted to be a small secreted Ig protein, we hypothesize that it serves a signaling function, similar to PDF neuropeptide, in the circadian circuit. In this R21 application, we propose to study the functions of NKT in the regulation of adult rhythmic behavior and sleep. Aim 1 will use conditional expression approaches to verify that NKT protein has a physiological requirement in the adult brain for the regulation of behavior. Additionally, it will delineate the neuronal requirements for the protein in sleep and circadian behavior. Aim 2 will examine genetic interactions with clock and sleep-altering mutations to better understand NKT's mechanistic functions in the relevant neuronal circuits. This aim will also ask whether NKT is secreted from neurons and test the hypothesis that the Ig protein is required for molecular oscillator function or synchronization of the clock neuron population. Proteins homologous to NKT, with single immunoglobulin domains are present in many species including C. elegans, Drosophila and humans (e.g., small secreted Neuregulin-2 isoforms). Thus, the research is likely to provide novel insights about signaling mechanisms that function in the differentiated brain.