Role of post-transcriptional gene regulation in differentiating retinal ganglion cells

神经元是一种特殊的细胞，它们通过被称为树突和轴突的过程相互交流。在它们的生长和分化过程中，神经元树突和轴突从细胞体伸出并延长，以响应营养因子的提示，直到它们到达它们的目标。这个过程被称为神经发生，对于在神经元和它们的靶点之间正确地建立功能和可行的连接是必不可少的。未能建立这种联系的神经元会导致细胞死亡。神经突起需要蛋白质、脂类和其他分子的持续供应，才能延长和正常发挥功能。这些分子通常是在细胞体中产生的，然后通过一个复杂的分子网络沿着轴突运输到突触。除了这种穿梭机制外，最近的研究表明，许多蛋白质实际上是在需要它们的时候和地点在树突中局部产生的。这一过程使神经元能够对特定刺激做出更快、更准确的反应。蛋白质是由信使核糖核酸或信使核糖核酸分子产生的，这些分子通常位于细胞核心或细胞核附近。最近，在神经突起的远端部分发现了mRNAs，在那里它们被翻译成蛋白质。调节mRNAs的稳定性和半衰期是细胞用来调节蛋白质合成的机制之一。在用于调节这一过程的几种手段中，microRNAs，一种非常短的RNA分子，已被发现发挥着非常重要的作用。通过与信使核糖核酸的特定部分结合，它们会引发信使核糖核酸的降解，从而间接减少特定蛋白质的产生。因此，在我们的实验方案中，我们希望研究亚细胞定位以及microRNAs在神经突起延长中所起的作用。此外，还将通过分析DICER的定位和表达来补充这一点，DICER是生产成熟microRNAs的关键酶之一。特别是，我们想了解Dier和microRNAs是否在伸长的轴突中重新定位，以及它们是否在这一过程中发挥了因果作用。这将使我们能够更好地了解蛋白质合成是如何在生长中的神经元中受到调节的，并通过实验调节迪特尔的活动，从而确定这一机制的任何损害是否会对神经细胞造成任何损害。此外，根据我们最近关于microRNAs调节BRN-3b转录因子mRNA的结果，我们想要分析在诱导视网膜神经节细胞(RGC)延长轴突时发生类似的机制。BRN-3b在RGC发育过程中表达，对它们的生存至关重要，缺乏BRN-3b的小鼠RGC损失高达70%就证明了这一点。因此，对BRN-3b在解释的RGC中转录后调控的分析将成为转录后调控在RGC中总体作用的范例。

Neurons are specialized cells which communicate with each other and other cells via processes called dendrites and axons. During their growth and differentiation, neuronal dendrites and axons protrude and elongate from the cell body in response to trophic factor cues until they reach their target. This process, called neuritogenesis, is essential for the proper establishment of functional and viable connections between neurons and their targets. Neurons that fail to establish such contact undergo cell death. Neurites need a constant supply of proteins, lipids and other molecules in order to elongate and function properly. These molecules are generally produced in the cell body and then transported along the neurite to the synapse by an elaborate network of molecules. Alongside this shuttling mechanism, it has been recently shown that many proteins are actually produced locally in the dendrite when and where they are needed. This process allows neurons to have a faster and more precise response to specific stimuli. Proteins are produced from molecules called messenger RNA or mRNA which are normally found close to the cellular core, or nucleus. Very recently mRNAs have been found in distal parts of neurites where they are translated into proteins. The regulation of mRNAs stability and half-life is one of the mechanisms used by cells to modulate protein synthesis. Amongst the several means used to regulate this process, microRNAs, very short RNA molecules, have been found to play a very important part. By binding to a specific part of the mRNA they trigger its degradation and thus indirectly reduce the production of specific proteins. In our experimental proposal we would thus like to study the sub-cellular localization and the role played by microRNAs in the elongation of neurites. This will be complemented by analyzing the localization and expression of Dicer, one of the key enzymes involved in the production of mature microRNAs. In particular we want to understand whether Dicer and microRNAs are relocated in elongating neurites and whether they play a causative role in this process. This will allow us to better understand how protein synthesis is regulated in growing neurons and by experimentally modulating Dicer's activity, whether any impairment of this mechanism can cause any damage to nerve cells. Furthermore, following our recent results on the regulation of the Brn-3b transcription factor mRNA by microRNAs, we would like to analyze the occurrence of a similar mechanism in retinal ganglion cells (RGC) induced to elongate neurites. Brn-3b is expressed during RGC development, and is essential for their survival as evidenced by the loss of up to 70% of RGC in mice devoid of Brn-3b. The analysis of Brn-3b post-transcriptional regulation in explanted RGC will be thus be a paradigm of the overall role of post-transcriptional regulation in RGC.