Sasaki Yukio, Gross Christina, Xing Lei, Goshima Yoshio, Bassell Gary J
Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, 30322; Department of Molecular Pharmacology and Neurobiology, Yokohama City University Graduate School of Medicine, Yokohama, 236-0004, Japan.
Dev Neurobiol. 2014 Mar;74(3):397-406. doi: 10.1002/dneu.22113. Epub 2013 Sep 13.
There is increasing evidence that localized mRNAs in axons and growth cones play an important role in axon extension and pathfinding via local translation. A few studies have revealed the presence of microRNAs (miRNAs) in axons, which may control local protein synthesis during axon development. However, so far, there has been no attempt to screen for axon-enriched miRNAs and to validate their possible localization to growth cones of developing axons from neurons of the central nervous system. In this study, the localization of miRNAs in axons and growth cones in cortical neurons was examined using a "neuron ball" culture method that is suitable to prepare axonal miRNAs with high yield and purity. Axonal miRNAs prepared from the neuron ball cultures of mouse cortical neurons were analyzed by quantitative real-time RT-PCR. Among 375 miRNAs that were analyzed, 105 miRNAs were detected in axons, and six miRNAs were significantly enriched in axonal fractions when compared with cell body fractions. Fluorescence in situ hybridization revealed that two axon-enriched miRNAs, miR-181a-1* and miR-532, localized as distinct granules in distal axons and growth cones. The association of these miRNAs with the RNA-induced silencing complex further supported their function to regulate mRNA levels or translation in the brain. These results suggest a mechanism to localize specific miRNAs to distal axons and growth cones, where they could be involved in local mRNA regulation. These findings provide new insight into the presence of axonal miRNAs and motivate further analysis of their function in local protein synthesis underlying axon guidance.
越来越多的证据表明，轴突和生长锥中的局部mRNA通过局部翻译在轴突延伸和路径寻找中发挥重要作用。一些研究已经揭示了轴突中存在微小RNA（miRNA），其可能在轴突发育过程中控制局部蛋白质合成。然而，到目前为止，尚未有人尝试筛选轴突富集的miRNA并验证它们可能定位于中枢神经系统神经元发育中的轴突生长锥。在本研究中，使用适合高产率和高纯度制备轴突miRNA的“神经元球”培养方法，检测了皮质神经元轴突和生长锥中miRNA的定位。通过定量实时RT-PCR分析从小鼠皮质神经元的神经元球培养物中制备的轴突miRNA。在分析的375种miRNA中，在轴突中检测到105种miRNA，与细胞体部分相比，有6种miRNA在轴突部分显著富集。荧光原位杂交显示，两种轴突富集的miRNA，miR-181a-1*和miR-532，定位于远端轴突和生长锥中的不同颗粒。这些miRNA与RNA诱导沉默复合体的关联进一步支持了它们在大脑中调节mRNA水平或翻译的功能。这些结果提示了一种将特定miRNA定位于远端轴突和生长锥的机制，在那里它们可能参与局部mRNA调节。这些发现为轴突miRNA的存在提供了新的见解，并激发了对其在轴突导向基础上的局部蛋白质合成中功能的进一步分析。
