Satiety signaling in Caenorhabditis elegans

描述(申请人提供)：我们建议研究线虫线虫食欲控制的分子机制。就像哺乳动物一样，线虫的食欲受到饥饿信号的促进，而受到饱腹感信号的抑制。这两种对食物可获得性的反应在行为上是相似的，在分子机制上与类似的哺乳动物行为重叠。基于这一保守性，我们的目标是在线虫中建立一个遗传模型系统来研究食欲控制。我们预计，分子机制将从根本上类似于哺乳动物的机制，但更简单，因此更容易解开。此外，我们希望，蠕虫中可用的强大遗传工具将允许快速阐明新的途径，这些途径与哺乳动物行为的相关性随后可以得到测试。我们广泛的长期目标是在分子水平上了解动物如何调节其食物摄入量。我们未来五年的目标是研究控制饱腹感的细胞和分子机制。我们将检验以下假设：1.ASI感觉神经元活动发出营养良好的信号，并激发饱腹感反应。2.ASI的作用是通过释放多肽和蛋白质激素实现的，包括胰岛素样多肽和转化生长因子-2样多肽DAF-7。3.饱腹感激素基因的表达随着营养状况的改善而增加。4.饱腹感荷尔蒙作用于下游神经元以改变行为。5.环GMP(CGMP)激活ASI和下游神经元中cGMP依赖的蛋白激酶(PKG)以产生饱足感。目的1.确定饱腹感的信号机制(假设1、4、5)。测定单虫饱足行为的时程。通过结合激光显微外科手术(假设2、4和5)，通过表达来自神经元特异性启动子的转基因来确定信号和受体基因在胰岛素、转化生长因子-2和cGMP途径中的作用位置和作用时间。通过上位性研究来确定信号作用的顺序，这些研究测量了功能缺失突变背景中过度表达和功能增益突变的影响。目的2.寻找与饱腹感相关的多肽和蛋白质激素基因(假设2、3)。利用基因芯片和定量RT-PCR技术，寻找其转录受饥饿和再摄食或cGMP和PKG调控的多肽基因。突变这些基因或下调它们的表达，以确定对饱腹感行为的影响。通过全基因组测序，筛选饱食诱导的静止缺陷突变基因。目的3.测定ASI的突触活性和多肽的释放(假设1，2)。开发通过对突触小泡pH传感器突触Hluorin进行光漂白后恢复荧光来测量突触活性的方法。开发测量神经元释放荧光肽的方法。使用这些方法来确定ASI活性和多肽释放如何对诱导饱腹感的条件做出反应。 与公共健康相关：肥胖是一个严重且日益严重的健康问题，其原因是吃得太多，无法满足人体的新陈代谢需求。更好地了解调节食欲的信号可能会带来更好的控制食物摄入量的方法，从而更好地控制肥胖。

DESCRIPTION (provided by applicant): We propose to investigate the molecular mechanisms of appetite control in the nematode Caenorhabditis elegans. Just as in mammals, C elegans appetite is promoted by hunger signals and suppressed by satiety signals. Both responses to food availability are similar behaviorally and overlap in molecular mechanism with analogous mammalian behaviors. Based on this conservation, we aim to establish in C elegans a genetic model system to study appetite control. We anticipate that molecular mechanisms will be fundamentally similar to those of mammals, but simpler and therefore easier to unravel. Furthermore, the powerful genetic tools available in the worm will, we hope, allow rapid elucidation of new pathways, whose relevance to mammalian behavior can subsequently be tested. Our broad long-term objective is to understand at a molecular level how an animal regulates its food intake. Our goal in the next five years is to investigate the cellular and molecular mechanisms that control satiety. We will test the following hypotheses: 1. ASI sensory neuron activity signals nutritional well-being and provokes a satiety response. 2. ASI's effects are mediated by the release of peptide and protein hormones, including insulin-like peptides and the TGF-2-like peptide DAF-7. 3. Expression of satiety hormone genes increases in response to nutritional well-being. 4. Satiety hormones act on downstream neurons to change behavior. 5. Cyclic GMP (cGMP) activates cGMP-dependent protein kinase (PKG) in ASI and in downstream neurons to produce satiety. Aim 1. Identify satiety signaling mechanisms (hypotheses 1, 4, 5). Determine the time-course of satiety behavior in single worms. Determine the sites and times of action of signal and receptor genes in the insulin, TGF-2, and cGMP pathways by expression of transgenes from neuron-specific promoters in combination with laser microsurgery (hypotheses 2, 4, and 5). Determine the order of signal action by epistasis studies measuring the effects of overexpression and gain-of-function mutations in loss-of-function mutant backgrounds. Aim 2. Find peptide and protein hormone genes whose transcription correlates with satiety (hypotheses 2, 3). Using microarrays and quantitative RT-PCR, find peptide genes whose transcription is regulated by starvation and refeeding, or by cGMP and PKG. Mutate these genes or knock down their expression and determine the effect on satiety behavior. Screen for mutants defective in satiety- induced quiescence and identify the mutated genes by whole-genome sequencing.Aim 3. Measure synaptic activity and release of peptides from ASI (hypotheses 1, 2). Develop methods to measure synaptic activity by recovery of fluorescence after photobleaching of the synaptic vesicle pH sensor synaptopHluorin. Develop methods to measure release of fluorescent peptides from neurons. Use these methods to determine how ASI activity and peptide release respond to conditions that induce satiety. PUBLIC HEALTH RELEVANCE: Obesity is a serious and growing health problem, brought on by eating more than is necessary to fulfill a person's metabolic needs. A better understanding of the signals that regulate appetite may lead to better methods for controlling food intake, and therefore to better control of obesity.