Identifying New Regulators of Leptin-Like Signaling in Drosophila Brain Neurons

描述（由申请人提供）：瘦素是一种控制脂肪储存和能量消耗的激素。瘦素由脂肪细胞产生，作用于调节能量平衡的下丘脑神经元。高瘦素水平是能量储存充足的指标，应该导致停止进食。然而，大多数肥胖的人都对瘦素有抵抗力。瘦素与JAK/STAT通路偶联受体相互作用，瘦素耐药可由该受体下游通路的下调引起。如果下丘脑神经元中瘦素受体信号的其他调节因子可以被确定，那么操纵它们的活动可能提供克服瘦素抵抗的方法。这一提议描述了一种利用果蝇遗传学找到这种调节因子的新方法。果蝇有专门的脂肪细胞，特定的大脑神经元组的活动控制脂肪的储存和代谢。本文报道的初步结果表明，果蝇的脂肪含量受到瘦素样JAK/STAT信号通路的调节，该信号通路作用于脂肪调节的“Fru”神经元。这些发现表明，果蝇和哺乳动物使用一些相似的遗传和神经机制来控制脂肪储存。果蝇是一种适合快速和廉价的前向遗传筛选的模型系统，我们的研究结果表明，在果蝇中发现的保守的JAK/STAT调节因子可能与哺乳动物系统的研究相关。第一个具体目标是通过调节Fru神经元中的JAK/STAT信号来寻找影响脂肪含量的蛋白质。这些将通过筛选一组约200个在培养细胞中被鉴定为JAK/STAT通路调节剂的基因来鉴定，其中约75%具有人类同源物或亲属。每个基因的表达将在Fru神经元中使用转基因RNAi敲除，敲除影响甘油三酯水平的基因将被选择用于进一步的研究。确定的候选基因将根据与人类基因的序列关系、效应大小和方向、可行的功能丧失突变的可用性和可用于标记的插入元件进行优先排序。第二个特定目标的目标是将特异性目标1中确定的调节剂的最高优先级子集置于其分子和细胞背景中。遗传上位分析将用于确定调节剂是作用于受体的上游还是下游，以及它们是否控制Fru神经元的放电。调节因子将在体内荧光标记，以揭示其细胞和亚细胞表达模式。同样的标记策略将用于创建“驱动”线，将赋予基因表达

DESCRIPTION (provided by applicant): Leptin is a hormone that controls fat storage and energy expenditure. Leptin is made by adipocytes and acts on hypothalamic neurons that regulate energy balance. High leptin levels are an indicator of sufficient energy stores and should cause cessation of eating. However, most obese humans are leptin resistant. Leptin interacts with a JAK/STAT pathway-coupled receptor, and leptin resistance can be caused by downregulation of the pathway downstream of the receptor. If additional regulators of leptin receptor signaling in hypothalamic neurons could be identified, manipulation of their activities might provide ways to overcome leptin resistance. This proposal describes a new way to find such regulators using Drosophila genetics. Drosophila has dedicated adipocytes, and the activities of specific sets of brain neurons control fat storage and metabolism. The preliminary results reported here show that Drosophila fat content is regulated by a leptin-like JAK/STAT signaling pathway that acts in fat-regulating 'Fru' neurons. These findings show that flies and mammals use some similar genetic and neural mechanisms for control of fat storage. Drosophila is a model system that is amenable to fast and inexpensive forward genetic screening, and our results suggest that conserved JAK/STAT regulators identified in Drosophila would have relevance for research in mammalian systems. The objective of the first specific aim is to find proteins that affect fat content by regulating JAK/STAT signaling in Fru neurons. These will be identified by screening a set of ~200 genes identified as modulators of the JAK/STAT pathway in cultured cells, about 75% of which have human orthologs or relatives. Expression of each gene will be knocked down in Fru neurons using transgenic RNAi, and those genes for which knockdown affects triglyceride levels will be selected for further study. The identified candidate genes will be prioritized based on sequence relationships with human genes, effect size and direction, and availability of viable loss-of- function mutations and inserted elements that can be used for tagging. The objective of the second specific aim is to place the highest-priority subset of the regulators identified in Specific Aim 1 into their molecular and cellular contexts. Analysis of genetic epistasis will be used to determine whether the regulators act upstream or downstream of the receptor, and if they control the firing of Fru neurons. Regulators will be fluorescently tagged in vivo to reveal their cellular and subcellular expression patterns. The same tagging strategy will be used to create 'driver' lines that will confer gene expression in neurons that normally express the regulator, allowing manipulation of their activities. The expected outcome of the research proposed in specific aims 1 and 2 is the definition of a set of conserved modulators of JAK/STAT signaling that function downstream of the receptor in fat-regulating neurons. Negative regulators identified in these experiments might be potential drug targets whose inhibition could upregulate anorexigenic leptin signaling in leptin-resistant obese individuals.