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

促进轴突再生的预处理因子的高通量测定

• 批准号: 9207488
• 负责人: Aaron Diantonio
• 金额: $ 53.43万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-15 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9207488
• 关键词: Adult; Afferent Neurons; Axon; Biological Assay; Development; Disease; Dissection; Drug Compounding; Gene Delivery; Genes; Genetic; Genetic study; Goals; Growth; Image; Image Analysis; In Vitro; Injury; Investigation; Laminin; Lead; Libraries; Liquid substance; Measurement; Methods; Microfluidics; Minor; Modeling; Molecular; Natural regeneration; Nerve Crush; Nerve Regeneration; Nervous System Trauma; Nervous system structure; Neuronal Injury; Neurons; Nociceptors; Open Reading Frames; Pathway interactions; Pattern; Peripheral Nervous System Diseases; Pharmaceutical Preparations; Phase; Preclinical Drug Evaluation; Process; Refractory; Regenerative response; Resistance; Robotics; Speed; Spinal Ganglia; Spinal cord injury; Subfamily lentivirinae; Therapeutic; Therapeutic Agents; Trauma; axon growth; axon regeneration; axonal degeneration; base; central nervous system injury; experience; experimental study; gene product; high throughput screening; improved; in vitro Assay; in vivo; injured; miniaturize; molecular marker; nerve injury; nervous system disorder; novel; novel strategies; novel therapeutic intervention; permissiveness; preconditioning; programs; public health relevance; regenerative; repaired; response; response to injury; sciatic nerve; screening; therapeutic candidate; therapeutic evaluation; tool

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.

描述（由申请人提供）：促进轴突再生的方法具有治疗受损和患病神经系统的巨大潜力。这种潜力在受损的CNS中最明显，例如在脊髓损伤中，其中基本上没有轴突再生。即使在外周，增加轴突再生的速度和程度也将提供重要的治疗益处。在这个建议中，我们概述了实验，以促进轴突再生的治疗调用预处理反应。预处理的分子基础知之甚少，但其在损伤后刺激轴突再生和增强轴突在非允许底物上生长的能力使其成为开发用于治疗受损神经系统的新方法的重要靶点。我们已经开发了一个完全在体外预处理试验在原代神经元，这将允许第一个高通量的药物和遗传学研究这一过程。我们计划使用高通量方法来识别预处理途径，这些方法是从我们开发的用于探索轴突变性的方法中改编的，从而能够快速筛选化合物和遗传途径。使用成年DRG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，我们将优化和优化筛选试验和图像分析（R21阶段），然后使用高通量筛选和成像分析，以确定化合物和/或基因，通过促进“预处理”反应增强轴突再生长（R33阶段）。其次，我们将开发二次筛选，以检测预处理的分子标志物，神经元亚型特异性预处理反应，以及基于微流体的抑制性基质（R21期）轴突生长检测。这些试验将用于进一步表征初步筛选（R33阶段）的“命中”。第三，我们将使用坐骨神经挤压试验来检查筛选中鉴定的几种优先化合物的体内活性（R33阶段）。通过这些实验，我们希望发现损伤诱导的预处理的试剂和途径，这将导致刺激强大的轴突再生和生长抑制底物的新方法，这将可能导致新的治疗受损的神经系统。