Exploring the role of microRNAs in injury-induced axonal growth in the CNS

描述(申请人提供)：在脊髓损伤中，轴突从神经元细胞体中被切断，轴突再生不足是导致中枢神经系统(CNS，包括大脑和脊髓)功能恢复不佳或有限的主要原因。虽然早期的研究强调了神经元-外部机制的重要性，但最近该领域的发展强调了神经元对中枢神经系统损伤后轴突生长和再生的内在控制的中心作用。与此同时，越来越清楚的是，一次瞄准一个分子或途径不太可能带来有功能意义的轴突生长和再生。MiRNAs是一种小的非编码RNA，在转录后调节蛋白质的合成。一个miRNA可以通过多种途径调节多种蛋白质的表达，有时在功能上与之相关。在这里，我们建议探讨miRNAs在中枢神经系统损伤后脊髓轴突萌发和再生中的作用。我们假设，在神经元中，一些miRNAs抑制生长，而另一些则促进生长。中枢神经系统损伤后抑制生长和促进生长的miRNAs的表达逆转，可能使神经元进入更多的再生状态，从而促进轴突的生长和再生。在目标1中，我们将系统地描述脊髓损伤前后皮质脊髓神经元和轴突中miRNA的表达，并将这些数据与出生后仍具有显著轴突生长能力的神经元中的miRNA表达进行比较。比较不同再生能力的神经元中miRNA的表达谱可能为揭示正向或负向调节轴突生长的miRNAs的模式和身份提供重要线索。表达谱分析将与靶标预测一起考虑，以缩小要进行功能测试的候选miRNAs的范围。在目标2中，我们将使用微流体室评估通过过表达或抑制体外培养的轴突生长来操纵候选miRNAs的效果。我们将使用腺相关病毒(AAV)来传递miRNAs或抑制性海绵结构。这些实验将帮助我们进一步缩小活体研究的候选数量。在目标3中，我们将评估操纵候选miRNAs对锥体切开术和脊髓背侧半横断损伤后皮质脊髓轴突萌发和再生的影响。总之，这些研究将开始评估miRNAs在促进中枢神经系统损伤后轴突修复方面的作用和治疗潜力。MiRNAs同时调控多个分子靶点和途径的独特特性使其成为治疗疾病和损伤的诱人靶点和工具。这一建议代表了将miRNA生物学应用于中枢神经系统损伤后轴突修复的早期步骤。

DESCRIPTION (provided by applicant): In spinal cord injuries, axons are cut from the neuronal cell bodies, and lack of axon regeneration is the principal cause of no or limited functional recovery in the central nervous system (CNS, including the brain and the spinal cord). While earlier studies emphasized the importance of neuron-extrinsic mechanisms, recent development in the field highlighted the central role of neuron-intrinsic control of axon growth and regeneration after CNS injury. Meanwhile, it is increasingly clear that targeting one molecule or pathway at a time is unlikely to bring about functionally meaningful axon growth and regeneration. miRNAs are small non-coding RNAs that post-transcriptionally regulate protein synthesis. One miRNA can regulate the expression of multiple proteins in multiple pathways, sometimes related in function. Here we propose to explore the role of miRNAs in spinal axon sprouting and regeneration after CNS injury. We hypothesize that in neurons some miRNAs are growth inhibitory while others are growth promoting. Reversal of the expression of growth inhibitory and promoting miRNAs after CNS injury may allow neurons to enter a more regenerative state, thus promoting axon growth and regeneration. In Aim 1, we will systematically profile miRNA expression in corticospinal neurons and axons before and after spinal cord injury and compare these data to miRNA expression in postnatal neurons that still possess significant axon growth ability. A comparison of miRNA expression profiles in neurons of different levels of regenerative abilities may provide important clues to the pattern and identiy of the miRNAs that may positively or negative regulate axon growth. The expression profiling will be considered together with target predictions to narrow down the candidate miRNAs to be functionally tested. In Aim 2, we will assess the effect of manipulating candidate miRNAs by overexpression or inhibition on axon growth in vitro using microfluidic chambers. We will use adeno-associated virus (AAV) to deliver miRNAs or inhibitory sponge constructs. These experiments will help us to further narrow down the number of candidates for in vivo studies. In Aim 3, we will assess the effect of manipulating candidate miRNAs on corticospinal axon sprouting and regeneration using pyramidotomy and dorsal hemisection spinal cord injury respectively. Together, these studies will start to assess the role and therapeutic potential of miRNAs for promoting axonal repair after CNS injury. The unique feature of miRNAs regulating multiple molecular targets and pathways simultaneously renders them attractive therapeutic targets and tools for diseases and injuries. This proposal represents an early step in the application of miRNA biology to axonal repair after CNS injury.