Regulation of growth cone guidance by localized microRNA

描述（由申请人提供）：mirna已成为一类功能强大的保守非编码rna，其转录后调节基因表达，并在神经元发育和突触功能的许多方面发挥关键作用。mirna的表达和/或功能受损与几种神经系统疾病有关。一个主要的空白是我们对特定的microrna在轴突生长锥上的定位，以及它们是否在调节生长锥运动和引导的局部蛋白质合成依赖作用中发挥重要作用的理解不足。本应用程序的目的是探索新的概念，即定位于生长锥的microRNAs （miRNAs）提供了一种受调节的分子机制，以影响线索介导的局部蛋白质合成和局部蛋白质合成依赖轴突指导。我们将测试生长锥microrna调节局部翻译以调节吸引力和排斥性转向的假设。对培养的皮质神经元进行定量荧光原位杂交将用于评估轴突部分富集的microrna可能的生长锥定位。我们提出了在培养的皮质神经元中可视化microrna定位的方法，在微流体装置中操纵轴突microrna和靶mrna翻译，以及在活神经元中用于局部翻译和生长锥引导的图像荧光报告。目的1将通过分析皮层神经元球轴突部分的microrna并使用荧光原位杂交素可视化其定位来鉴定定位于轴突的microrna。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信号的转向反应的影响。目的3将利用荧光报告者的免疫荧光和活细胞成像技术，研究选定的候选microrna对生长锥轴突蛋白表达和局部蛋白合成的影响。本研究将促进我们对生长锥定位mirna的有限理解，并阐明它们在生长锥指导下局部蛋白质合成中的机制作用。本研究有望激发研究轴突microrna在体内神经系统发育中的功能，以及microrna调节功能障碍导致神经系统疾病的研究。这些研究对未来在神经系统疾病治疗中调节microRNA表达或功能以操纵轴突生长和连通性的治疗策略具有重要意义。

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.