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，应该引起停止进食。然而，大多数肥胖的人是瘦素抵抗。瘦素与JAK/STAT途径偶联受体相互作用，并且瘦素抗性可由受体下游途径的下调引起。如果能确定下丘脑神经元中瘦素受体信号传导的其他调节剂，则操纵它们的活性可能提供克服瘦素抵抗的方法。该提案描述了一种利用果蝇遗传学寻找此类调节因子的新方法。果蝇有专门的脂肪细胞，特定的脑神经元组的活动控制脂肪储存和代谢。这里报告的初步结果表明，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“Fru”神经元。这些发现表明，苍蝇和哺乳动物使用一些类似的遗传和神经机制来控制脂肪储存。果蝇是一个模型系统，这是适合快速和廉价的正向遗传筛选，我们的研究结果表明，保守的JAK/STAT调节在果蝇中确定的哺乳动物系统的研究将有相关性。第一个具体目标的目的是找到通过调节Fru神经元中的JAK/STAT信号传导来影响脂肪含量的蛋白质。这些将通过筛选培养细胞中被鉴定为JAK/STAT通路调节剂的一组约200个基因来鉴定，其中约75%具有人类直系同源物或亲属。将使用转基因RNAi在Fru神经元中敲低每个基因的表达，并且将选择敲低影响甘油三酯水平的那些基因用于进一步研究。将根据与人类基因的序列关系、效应大小和方向以及可行的功能丧失突变和可用于标记的插入元件的可用性，对所识别的候选基因进行优先级排序。第二个具体目标的目的是将具体目标1中确定的最高优先级的调节子子集置于其分子和细胞背景中。遗传上位性的分析将用于确定调节剂是否作用于受体的上游或下游，以及它们是否控制Fru神经元的放电。调节因子将在体内被荧光标记，以揭示其细胞和亚细胞表达模式。相同的标记策略将用于创建“驱动”线，该驱动线将赋予基因表达， 神经元通常表达调节器，允许操纵它们的活动。具体目标1和2中提出的研究的预期结果是定义一组JAK/STAT信号传导的保守调节剂，其在脂肪调节神经元中的受体下游发挥作用。在这些实验中发现的负调节因子可能是潜在的药物靶点，其抑制可以上调瘦素抵抗性肥胖个体中的促瘦素信号传导。

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.