A HIGH-THROUGHPUT ASSAY FOR PRECONDITIONING FACTORS THAT PROMOTE AXONAL REGENERAT

描述(申请人提供)：促进轴突再生的方法在治疗受伤和患病的神经系统方面具有巨大的潜力。这种潜力在受损的中枢神经系统中最为明显，例如在脊髓损伤中，基本上没有轴突再生。即使在外周，提高轴突再生的速度和程度也会提供重要的治疗益处。在这项提案中，我们概述了通过治疗性地调用预适应反应来促进轴突再生的实验。预适应的分子基础尚不清楚，但其刺激损伤后轴突再生和促进非许可基质上的轴突生长的能力使其成为开发治疗受损神经系统的新方法的重要靶点。我们已经在原代神经元中开发了一种完全体外预适应试验，这将使这一过程的第一次高通量药物和遗传学研究成为可能。我们计划使用高通量方法识别预适应途径，这些方法来自于我们开发的探索轴突变性的方法，这些方法能够快速筛选化合物和遗传途径。利用成年背根节神经元，我们计划筛选药物化合物文库和慢病毒开放阅读框架(ORF)文库。首先，我们将优化和微型化筛选分析和图像分析(R21阶段)，然后使用高通量筛选和图像分析来识别通过促进‘预适应’反应促进轴突再生长的化合物和/或基因(R33阶段)。其次，我们将开发二次筛选来检测预适应的分子标志物、神经元亚型特异性的预适应反应，以及基于微流体的抑制底物上轴突生长的检测(R21期)。这些检测将被用来进一步表征来自主屏幕(R33阶段)的“命中”。第三，我们将使用坐骨神经挤压试验来检查在筛选(R33阶段)中确定的几个优先化合物的体内活性。通过这些实验，我们希望发现损伤诱导的预适应的试剂和途径，这将导致新的方法来刺激强大的轴突再生和在抑制底物上的生长，这将可能导致对受损神经系统的新治疗。

DESCRIPTION (provided by applicant): Methods to promote axonal regeneration have tremendous potential to treat the injured and diseased nervous system. This potential is most clear in the injured CNS, such as in spinal cord injury, where there is essentially no axon regeneration. Even in the periphery, increasing the speed and extent of axonal regeneration would provide important therapeutic benefits. In this proposal, we outline experiments to promote axon regeneration by therapeutically invoking the preconditioning response. The molecular basis of preconditioning is poorly understood but its ability to stimulate axonal regeneration after injury and to enhance axonal growth over non-permissive substrates makes it an important target for development of new approaches for treating the damaged nervous system. We have developed a fully in vitro preconditioning assay in primary neurons, which will allow for the first high throughput drug and genetic studies of this process. We plan to identify preconditioning pathways using high-throughput methods adapted from those we developed to explore axonal degeneration that enable rapid screening of compounds and genetic pathways. Using adult DRG neurons we plan to screen libraries of drug compounds and lentivirus open reading frame (ORF) libraries. First, we will optimize and miniaturize the screening assay and image analysis (R21 phase) and then use high-throughput screening and imaging analysis to identify compounds and/or genes that enhance axon re-growth by promoting a 'preconditioning' response (R33 phase). Second, we will develop secondary screens to assay molecular markers of preconditioning, neuronal sub-type-specific preconditioning responses, and a microfluidics based assay for axonal growth on inhibitory substrates (R21 phase). These assays will be used to further characterize 'hits' from the primary screens (R33 phase). Third, we will use a sciatic nerve crush assay to examine the in vivo activity of a few prioritized compounds identified in the screens (R33 phase). Through these experiments we hope to uncover agents and pathways of injury-induced preconditioning that will lead to new methods for stimulating robust axonal regrowth and growth on inhibitory substrates that will potentially lead to new treatments for the damaged nervous system.