Regulation of growth cone guidance by localized microRNA

局部 microRNA 对生长锥引导的调节

• 批准号: 8501908
• 负责人: GARY J BASSELL
• 金额: $ 23.4万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8501908
• 关键词: Adaptor Signaling Protein; Address; Adhesions; Axon; Behavior; Binding Proteins; Cues; Dendrites; Dendritic Spines; Disease; FMRP; Fluorescent in Situ Hybridization; Functional disorder; Future; Gene Expression; Genetic Translation; Genets; Goals; Growth Cones; Image; Imagery; Immunofluorescence Immunologic; In Situ; Life; Mediating; Messenger RNA; MicroRNAs; Microfluidic Microchips; Microtubules; Modeling; Molecular; Morphogenesis; Morphology; Motor; Neurologic; Neurons; Play; Protein Biosynthesis; Proteins; RNA-Binding Proteins; Receptor Signaling; Regulation; Reporter; Research; Role; Signal Transduction; Surface; Synaptic Receptors; Testing; Therapeutic; Translations; Untranslated RNA; Vertebral column; Work; axon growth; axon guidance; axon regeneration; cell motility; cellular imaging; in vivo; nervous system development; nervous system disorder; neuron development; protein expression; public health relevance; receptor; response; synaptic function

DESCRIPTION (provided by applicant): MiRNAs have emerged as a powerful class of conserved noncoding RNAs that regulate gene expression post- transcriptionally and play critical roles in numerous aspects of neuronal development and synaptic function. Impaired expression and/or function of miRNAs is implicated in several neurological diseases. A major gap is our poor understanding on the localization of specific microRNAs to the axon growth cone, and whether they play important roles to regulate local protein synthesis dependent effects on growth cone motility and guidance. The objectives of this application are to explore the new concept that microRNAs (miRNAs) localized to growth cones provide a regulated molecular mechanism to influence local protein synthesis underlying cue mediated and local protein synthesis dependent axon guidance. We will test the hypothesis that growth cone microRNAs regulate local translation to modulate attractive versus repulsive steering. Quantitative fluorescent in situ hybridization on cultured cortical neurons will be used to assess possible growth cone localization for microRNAs enriched in axonal fractions. We propose approaches to visualize the localization of microRNAs in cultured cortical neurons, to manipulate axonal microRNAs and target mRNAs translation in microfluidic devices, and image fluorescent reporters for local translation and growth cone guidance in live neurons. Aim 1 will identify miRNAs that are localized to axons by profiling microRNAs from axonal fractions of cortical neuron balls and visualization of their localization using fluorescent in situ hybridizatin. Aim 2 will examine the effects of manipulating axonal microRNA levels and function on axon outgrowth, growth cone morphology and steering responses to attractive and repulsive cues. Aim 3 will examine the effects of select candidate microRNAs on axonal protein expression and local protein synthesis in growth cones using immunofluorescence and live cell imaging of fluorescent reporters. The proposed research will advance our limited understanding of growth cone localized miRNAs and elucidate their mechanistic role in local protein synthesis underlying growth cone guidance. This research is expected to motivate studies to investigate the function of axonal microRNAs in the development of the nervous system in vivo, as well as dysfunction of micoRNA regulation leading to neurological diseases. These studies have important implications for future therapeutic strategies to modulate microRNA expression or function to manipulate axonal growth and connectivity in the treatment of neurologic disorders.

描述（由申请人提供）：MiRNA已经作为一类强大的保守非编码RNA出现，其在转录后调节基因表达，并且在神经元发育和突触功能的许多方面发挥关键作用。miRNAs的表达和/或功能受损与几种神经系统疾病有关。一个主要的差距是我们对特定microRNA在轴突生长锥中的定位以及它们是否在调节生长锥运动和指导的局部蛋白质合成依赖性效应中发挥重要作用的理解不足。本申请的目的是探索新的概念，即定位于生长锥的microRNA（miRNAs）提供调节的分子机制以影响潜在的线索介导的和局部蛋白质合成依赖性轴突引导的局部蛋白质合成。我们将测试生长锥microRNA调节本地翻译，以调节吸引与排斥转向的假设。对培养的皮层神经元进行定量荧光原位杂交，以评估轴突组分中富集的microRNA可能的生长锥定位。我们提出的方法来可视化的microRNA在培养的皮层神经元的本地化，操纵轴突microRNA和靶mRNA的翻译在微流体设备，和图像荧光报告本地翻译和生长锥活神经元的指导。目的1将通过对来自皮层神经元球的轴突片段的microRNA进行分析并使用荧光原位杂交对其定位进行可视化来鉴定定位于轴突的miRNAs。目的2将研究操纵轴突microRNA水平和功能对轴突生长，生长锥形态和对吸引和排斥线索的转向反应的影响。目的3将使用荧光报告分子的免疫荧光和活细胞成像来检查选择的候选microRNA对轴突蛋白表达和生长锥中的局部蛋白合成的影响。这项研究将促进我们对生长锥定位的miRNAs的有限理解，并阐明它们在生长锥指导下的局部蛋白质合成中的机制作用。这项研究有望推动研究轴突microRNA在体内神经系统发育中的功能，以及导致神经系统疾病的microRNA调节功能障碍。这些研究对未来的治疗策略具有重要意义，以调节microRNA的表达或功能，从而在神经系统疾病的治疗中操纵轴突生长和连接。