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神经系统损伤和病变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，如脊髓损伤，基本上没有轴突再生。即使在外周，增加轴突再生的速度和范围将提供重要的治疗效益。在本提案中，我们概述了通过治疗性地调用预处理反应来促进轴突再生的实验。预适应的分子基础尚不清楚，但它在损伤后刺激轴突再生和促进轴突在非允许底物上生长的能力使其成为开发治疗受损神经系统新方法的重要目标。我们已经在初级神经元中开发了一种完全体外预处理试验，这将允许对这一过程进行首次高通量药物和遗传研究。我们计划使用高通量方法来识别预处理途径，这些方法改编自我们开发的用于探索轴突变性的方法，从而能够快速筛选化合物和遗传途径。利用成体DRG神经元筛选药物化合物文库和慢病毒开放阅读框（ORF）文库。首先，我们将优化和小型化筛选实验和图像分析（R21期），然后使用高通量筛选和成像分析来识别通过促进“预处理”反应来增强轴突再生的化合物和/或基因（R33期）。其次，我们将开发二级筛选来检测预处理的分子标记，神经元亚型特异性预处理反应，以及基于微流体的抑制底物轴突生长检测（R21期）。这些检测将用于进一步表征来自主筛选的“命中”（R33期）。第三，我们将使用坐骨神经挤压试验来检查筛选中确定的一些优先化合物的体内活性（R33期）。通过这些实验，我们希望揭示损伤诱导的预适应的因子和途径，这将导致刺激强大的轴突再生和抑制性底物生长的新方法，这将有可能导致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.