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

• 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
• 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; 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

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.

项目摘要： 睡眠是一种广泛保守的行为，已知它受基因表达变化的调节。 但是，调节睡眠的分子基础仍然很少了解。我们实验室的研究 在其他地方，支持microRNA（mirs）参与的观念。 miR是短的非编码RNA 靶向特定mRNA的转录本（长度为20-24 bp），下调其表达。结果 遗传屏幕通过特异性结合的转基因表达下调miR。 特定的miRS（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基因突变都引起了急剧变化 果蝇的睡眠行为，包括减少和分散的总睡眠以及睡眠不足 稳态。使用MiR-190-Sp在不同大脑区域中有限数量的MiR-190-Sp表达使用 GAL4/UAS系统表明，miR-190功能的破坏必须在大量神经元中发生 影响果蝇睡眠调节。在分子水平上，我们的初步数据来自成人头的RNA SEQ 表明miR-190-SP的泛神经表达诱导多个基因的上调或下调 包括9个密切涉及多巴胺（DA）信号的基因 苍蝇。 miR-190-sp的时间控制的表达表明，完整的miR-190敲低 表型需要在化合过程中表达。仅在早期减少miR-190 发育阶段没有产生表型。成人特异性表达最小影响的睡眠时间，但 破坏的体内平衡。综上所述，我们的数据表明MiR-190在开发过程中的功能 指定成人唤醒系统的活性，并在成人睡眠体内稳态中持续作用。这 提案旨在揭示Mir-190在成人建立中的发展作用 睡眠行为，剖析多巴胺能信号的参与。 睡眠，发现MiR-190在体内平衡中的作用的细胞基因座。首先，我将确定 通过执行AGO-IP RNASEQ的miR-190的分子靶标，并通过行为验证这些靶标 实验。此外，我将追求miR-190成人表型的基础及其根据 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 确定其睡眠表型是否是由细胞数量或连通性变化引起的。通过遗传 编码的功能性Calexa成像传感器I将查看MiR-190 Hypomorph Flies中DA细胞的活性 并确定与对照苍蝇相比它们是否具有多动。最后，我将以Mir-190的特征 睡眠稳态调节表达的时空表达模式，通过表达miR-190海绵 已知会影响睡眠体内平衡和设计miR-190传感器的大脑区域，能够读取mir- 190级。这些实验将阐明mir在调节睡眠中的作用，这将是至关重要的 以了解正常睡眠以及病理睡眠状况。