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

鉴定果蝇脑神经元中瘦素样信号传导的新调节因子

• 批准号: 8563793
• 负责人: KAI G ZINN
• 金额: $ 24.98万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8563793
• 关键词: Adipocytes; Affect; Anti-Obesity Agents; Biological Models; Body Weight; Brain; Candidate Disease Gene; Cells; Chronic Disease; Cultured Cells; Disease; Down-Regulation; Drosophila genus; Drug Targeting; Eating; Elements; Energy Metabolism; Fatty acid glycerol esters; Gene Expression; Generic Drugs; Genes; Genetic; Genetic Epistasis; Genetic Screening; Goals; Hormones; Human; Hypothalamic structure; Incidence; Individual; Insertion Mutation; Leptin; Leptin resistance; Mammals; Maps; Metabolism; Molecular; Neurons; Obesity; Orthologous Gene; Outcomes Research; Pathway interactions; Pattern; Proteins; Public Health; Publishing; RNA Interference; Receptor Signaling; Relative (related person); Reporting; Research; Resistance; Signal Pathway; Signal Transduction; Site; System; Therapeutic; Transgenic Organisms; Triglycerides; Work; base; drug development; energy balance; feeding; fly; genetic regulatory protein; in vivo; insight; knock-down; leptin receptor; lipid metabolism; loss of function mutation; neuromechanism; obesity management; public health relevance; receptor; receptor coupling; research study; screening

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.

描述(申请人提供)：瘦素是一种控制脂肪储存和能量消耗的激素。瘦素是由脂肪细胞制造的，作用于调节能量平衡的下丘脑神经元。高水平的瘦素是能量储备充足的指标，应该会导致停止进食。然而，大多数肥胖的人对瘦素有抵抗力。瘦素与JAK/STAT途径偶联的受体相互作用，通过下调该受体下游的途径可引起瘦素抵抗。如果能够确定下丘脑神经元中瘦素受体信号的其他调节因子，那么操纵它们的活动可能会提供克服瘦素抵抗的方法。这项提议描述了一种利用果蝇遗传学来寻找这样的调节器的新方法。果蝇有专门的脂肪细胞，特定脑神经元的活动控制脂肪的储存和新陈代谢。这里报道的初步结果表明，果蝇的脂肪含量受瘦素样JAK/STAT信号通路的调节，该信号通路作用于脂肪调节的‘FEU’神经元。这些发现表明，苍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、廉价的正向遗传筛查的模式系统，我们的结果表明，在果蝇中发现的保守的JAK/STAT调节因子将与哺乳动物系统的研究相关。第一个特定目标的目的是找到通过调节Fru神经元中的JAK/STAT信号来影响脂肪含量的蛋白质。这些基因将通过在培养细胞中筛选一组被鉴定为JAK/STAT途径调节器的约200个基因来鉴定，其中约75%具有人类同源或近亲。每个基因的表达都将通过转基因RNAi在Fru神经元中被下调，而那些被下调影响甘油三酯水平的基因将被挑选出来进行进一步的研究。确定的候选基因将根据与人类基因的序列关系、影响大小和方向以及可用于标记的可行功能丧失突变和插入元件的可用性进行优先排序。第二个特定目标的目标是将特定目标1中确定的最优先的调节子集置于其分子和细胞环境中。对遗传上位性的分析将被用来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。调控子将在体内被荧光标记，以揭示它们的细胞和亚细胞表达模式。同样的标记策略将被用来创建将赋予基因在 正常情况下表达调节器的神经元，允许操纵其活动。在特定目标1和2中提出的这项研究的预期结果是定义了一组保守的JAK/STAT信号调节器，它们在脂肪调节神经元的受体下游发挥作用。在这些实验中发现的负调控因子可能是潜在的药物靶点，其抑制可以上调瘦素抵抗肥胖者的厌食性瘦素信号。