Unraveling miR-190 and its Role in Sleep in Drosophila melanogaster

揭示 miR-190 及其在果蝇睡眠中的作用

基本信息

批准号：
9988251
负责人：
Emmanuel J. Rivera-Rodriguez
金额：
$ 3.16万
依托单位：
BRANDEIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-12-16 至 2021-12-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9988251
关键词：
Acute Adult Affect Area Arousal Behavior Behavioral Binding Brain Brain region Cell Count Cells Cognition Disorders Control Animal Data Development Diabetes Mellitus Disease Dopamine Down-Regulation Drosophila genus Drosophila melanogaster Gene Expression Genes Genetic Genetic Screening Genetic Transcription Genotype Head Health Homeostasis Hour Hyperactivity Image Immunohistochemistry Immunoprecipitation Knowledge Length Light Link Maps Measures Messenger RNA MicroRNAs Molecular Molecular Target Morphology Mutation Neurons Obesity Pathologic Pattern Phenotype Physiological Porifera Process Pupa Reading Regulation Research Role Signal Pathway Signal Transduction Sleep Sleep Deprivation Sleep Disorders Sleep disturbances Societies System Time To specify Transcript Untranslated RNA Work base comorbidity cost critical period design dopaminergic neuron experimental study fly improved knock-down neuron development overexpression response sensor sleep behavior sleep regulation spatiotemporal therapeutic target tool transcriptome sequencing transgene expression

项目摘要

Project Summary: Sleep is a widely conserved behavior and it is known to be regulated by changes in gene expression. However, the molecular basis of the regulation of sleep remains poorly understood. Research from our lab, and elsewhere, supports the idea that microRNAs (miRs) are involved. miRs are short non-coding RNA transcripts (20-24 bp in length) that target specific mRNAs, downregulating their expression. Results from a genetic screen in which miRs were downregulated by expression of transgenes which specifically bind particular miRs (miR-SPs), demonstrated that miR-190 is involved in Drosophila sleep regulation. Pan- neuronal expression of miR-190-SP or mutation of the miR-190 gene both elicited dramatic changes in Drosophila sleep behavior, including decreased and fragmented total sleep, as well as deficient sleep homeostasis. Expression of miR-190-SP in limited numbers of cells in different brain regions using the Gal4/UAS system showed that disruption of miR-190 function must occur in a large number of neurons to affect Drosophila sleep regulation. At the molecular level, our preliminary data from RNA seq of adult heads showed that pan-neuronal expression of miR-190-SP induces an up or downregulation of multiple genes, including 9 genes which are intimately involved in dopamine (DA) signaling, the major pro-arousal system of the fly. Temporally-controlled expression of miR-190-SP demonstrated that the full miR-190 knockdown phenotype requires expression during the middle of pupation; reduction of miR-190 only in earlier developmental stages produced no phenotype. Adult-specific expression minimally affected sleep time, but disrupted homeostasis. Taken together, our data suggest that miR-190 functions during development to specify the activity of the adult arousal system and has an on-going role in adult sleep homeostasis. This proposal aims to unravel the developmental role of miR-190 in the establishment of adult sleep behaviors, dissect the involvement of dopaminergic signaling in this regulation of sleep, and discover the cellular locus of miR-190's role in homeostasis. First, I will identify the molecular targets of miR-190 by performing of Ago-IP RNAseq and validate those by means of behavioral experiments. Additionally, I will pursue the bases of miR-190 adult phenotype and its regulation by dopaminergic signaling. I will study the morphology of DA cells in these miR-190-deficient genotypes to determine if their sleep phenotype is caused by changes in cell number or connectivity. By using genetically encoded functional CaLexA imaging sensor I will look at the activity of DA cells in miR-190 hypomorph flies and determine if they are hyperactive, compared to control flies. Lastly, I will characterize miR-190's spatiotemporal pattern of expression for sleep homeostasis regulation by expressing a miR-190 sponge in brain areas known to affect sleep homeostasis and designing a miR-190 sensor capable of reading out miR- 190 levels. These experiments will shine light on the role of miRs in modulation of sleep and will be critical for the understanding of normal sleep, as well as pathological sleep conditions.

项目概要：睡眠是一种广泛保守的行为，已知它受到基因表达变化的调节。然而，人们对睡眠调节的分子基础仍然知之甚少。我们实验室的研究，和其他地方，支持 microRNA (miR) 参与的观点。 miR 是短的非编码 RNA 转录本（长度为 20-24 bp）靶向特定 mRNA，下调其表达。结果来自基因筛选，其中 miR 通过特异性结合的转基因表达下调特别是 miR（miR-SP），证明 miR-190 参与果蝇睡眠调节。平底锅- miR-190-SP 的神经元表达或 miR-190 基因的突变都会引起果蝇睡眠行为，包括总睡眠减少和碎片化，以及睡眠不足体内平衡。使用 miR-190-SP 在不同大脑区域的有限数量的细胞中表达 Gal4/UAS系统表明，miR-190功能的破坏必须发生在大量神经元中才能发挥作用。影响果蝇的睡眠调节。在分子水平上，我们来自成人头部 RNA seq 的初步数据显示 miR-190-SP 的泛神经元表达诱导多个基因的上调或下调，包括与多巴胺 (DA) 信号传导密切相关的 9 个基因，多巴胺信号传导是主要的促觉醒系统苍蝇的。 miR-190-SP 的时间控制表达表明，miR-190 完全敲低表型需要在化蛹中期表达； miR-190仅在早期减少发育阶段不产生表型。成人特异性表达对睡眠时间的影响最小，但体内平衡被破坏。综上所述，我们的数据表明 miR-190 在发育过程中发挥作用指定成人唤醒系统的活动，并在成人睡眠稳态中发挥持续作用。这该提案旨在揭示 miR-190 在成人建立中的发育作用睡眠行为，剖析多巴胺能信号在这种调节中的参与睡眠，并发现 miR-190 在体内平衡中的作用的细胞位点。首先，我将确定通过执行 Ago-IP RNAseq 确定 miR-190 的分子靶点，并通过行为学方法验证这些靶点实验。此外，我将研究 miR-190 成体表型的基础及其调控多巴胺能信号传导。我将研究这些 miR-190 缺陷基因型中 DA 细胞的形态确定他们的睡眠表型是否是由细胞数量或连接性的变化引起的。通过利用基因编码功能性 CaLexA 成像传感器我将观察 miR-190 亚型果蝇中 DA 细胞的活性并确定与对照果蝇相比，它们是否过度活跃。最后，我将描述 miR-190 的特征通过表达 miR-190 海绵来调节睡眠稳态的时空表达模式已知影响睡眠稳态的大脑区域并设计了能够读取 miR-的 miR-190 传感器 190 个级别。这些实验将揭示 miR 在调节睡眠中的作用，并且至关重要了解正常睡眠以及病理性睡眠状况。