Regulation of axon outgrowth by retrograde Ret signaling

通过逆行 Ret 信号调节轴突生长

• 批准号: 10364762
• 负责人: Alex Nechiporuk
• 金额: $ 36.57万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10364762
• 关键词: Actins; Address; Afferent Neurons; Animals; Axon; Axonal Transport; Behavior; Binding; Biochemistry; Biological; Biological Assay; Cell Adhesion; Cell Nucleus; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Data; Defect; Development; Distant; Dynein ATPase; Environment; Failure; Filopodia; Genes; Genetic Transcription; Goals; Growth; Growth Cones; Image; Individual; Injury; Knowledge; Lead; Length; Ligands; Link; Mediating; Molecular; Motor; Natural regeneration; Nature; Nerve Growth Factor Receptors; Nervous system structure; Neurons; Presynaptic Terminals; Primordium; Process; Receptor Activation; Regulation; Role; Scaffolding Protein; Sensory; Sensory Ganglia; Signal Transduction; System; Techniques; Testing; Vesicle; Work; Zebrafish; axon growth; base; differential expression; experimental study; genetic analysis; genetic approach; glial cell-line derived neurotrophic factor; imaging approach; in vivo; innovation; lateral line; mutant; nervous system development; neurotrophic factor; overexpression; programs; receptor; receptor internalization; response; retrograde transport; transcription factor; transcriptome; transcriptome sequencing; zebrafish development

During development or following injury, axon terminals must navigate through a complex environment to form functional connections. Ret is a neurotrophin receptor which is present at the tips of growing axons. Ret activation and internalization induces retrograde transport of the activated receptor to the cell body triggering a transcriptional response, which in turn promotes axon outgrowth. Despite its essential role during nervous system development, it is not known how activated Ret receptor is trafficked in axons, the nature of the transcriptional response induced by the retrograde Ret signaling, and how these transcriptional targets ultimately promote axon growth. To address these questions, we are using the unique advantages of zebrafish, including live imaging and genetic approaches, to identify the molecular mechanisms that govern retrograde transport of a neurotrophin receptor Ret and the subsequent transcriptional response. In our preliminary studies, we discovered that retrograde transport of Ret depends on binding to the scaffold protein, Jip3, which links cargo to the retrograde motor for transport. Both ret and jip3 mutants display truncated sensory axons, and live imaging revealed abnormal growth cone dynamics and reduced axon terminal elaboration. Using RNA sequencing, we identified a number of factors that are transcriptionally induced in response to Ret-Jip3 retrograde signaling and are putative regulators of actin-based protrusive behavior in this context. Based on this data, we hypothesize that Jip3-dependent retrograde transport of Ret induces factors that promote growth cone dynamics required for sensory axon extension. We will test this hypothesis in the three specific aims. In Aim 1, we will define the molecular mechanisms of Ret retrograde transport and the role of Jip3 in this process. Experiments in the second aim will define the defects in growth cone dynamics that lead to the failure of axon extension in ret and jip3 mutants and determine the role of the Ret retrograde signaling in this process. The last aim will investigate the transcriptional response elicited by the retrograde Ret signaling and how factors that are regulated by this transcriptional program promote growth cone dynamics. Altogether, our study combines innovative assays in zebrafish with in vivo techniques to determine the specific role of retrograde neurotrophin signaling in axon outgrowth. Our work will further uncover the mechanisms by which long-range neurotrophin signals are transduced into cellular responses that regulate growth cone dynamics and axon extension.

在发育期间或受伤后，轴突末端必须穿过复杂的环境以形成 功能连接。 Ret 是一种神经营养蛋白受体，存在于生长轴突的尖端。雷特 激活和内化诱导激活的受体逆行转运至细胞体，从而触发 转录反应，进而促进轴突生长。尽管它在紧张期间发挥着重要作用 系统开发时，尚不清楚激活的 Ret 受体如何在轴突中运输，其性质 逆行 Ret 信号诱导的转录反应，以及这些转录目标如何 最终促进轴突生长。为了解决这些问题，我们利用斑马鱼的独特优势， 包括实时成像和遗传方法，以确定控制逆行的分子机制 神经营养蛋白受体 Ret 的转运和随后的转录反应。在我们的初步 研究中，我们发现 Ret 的逆行转运依赖于与支架蛋白 Jip3 的结合，该蛋白 将货物连接到逆行电机进行运输。 ret 和 jip3 突变体都显示出截短的感觉轴突， 实时成像显示生长锥动力学异常和轴突末端精细化减少。使用RNA 通过测序，我们确定了许多响应 Ret-Jip3 转录诱导的因子 逆行信号传导，并且是在这种情况下基于肌动蛋白的突出行为的假定调节剂。基于 根据这些数据，我们假设 Jip3 依赖性 Ret 逆行转运诱导了促进生长的因子 感觉轴突延伸所需的锥体动力学。我们将在三个具体目标中检验这一假设。在 目标1，我们将定义Ret逆行转运的分子机制以及Jip3在此过程中的作用。 第二个目标的实验将确定导致轴突失败的生长锥动力学缺陷 ret 和 jip3 突变体中的延伸并确定 Ret 逆行信号在该过程中的作用。这 最后一个目标是研究逆行 Ret 信号引发的转录反应以及如何影响转录反应 受该转录程序调节的细胞可促进生长锥动态。总而言之，我们的研究 将斑马鱼的创新检测与体内技术相结合，以确定逆行的具体作用 轴突生长中的神经营养蛋白信号传导。我们的工作将进一步揭示长期影响的机制 神经营养蛋白信号被转化为调节生长锥动力学和轴突的细胞反应 扩大。