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

基本信息

批准号：
8798703
负责人：
Aaron Diantonio
金额：
$ 20.9万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-02-15 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8798703
关键词：
Adult Afferent Neurons Axon Biological Assay Development Disease Dissection Drug Compounding Gene Delivery Genes Genetic Genetic study Goals Growth Health 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 Proteins Refractory Regenerative response Resistance Robotics Speed Spinal Ganglia Spinal cord injury Subfamily lentivirinae Testing Therapeutic Therapeutic Agents Trauma axon growth axon regeneration axonal degeneration base experience high throughput screening improved in vitro Assay in vivo injured miniaturize molecular marker nerve injury novel novel strategies novel therapeutic intervention preconditioning programs regenerative repaired research study response response to injury sciatic nerve screening tool

项目摘要

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伤和患病的神经系统的巨大潜力。在受伤的中枢神经系统中，这种潜力最明显，例如在基本没有轴突再生的脊髓损伤中。即使在外围，增加轴突再生的速度和程度也将提供重要的治疗益处。在此提案中，我们概述了通过治疗方法调用预处理反应来促进轴突再生的实验。预处理的分子基础知之甚少，但其刺激受伤后的轴突再生并增强轴突生长的能力在非腐烂的底物上增强了轴突生长，这使其成为开发新方法治疗受损神经系统的重要目标。我们已经在原发性神经元中开发了完全体外的预处理测定法，这将允许对此过程进行首次高通量药物和遗传研究。我们计划使用我们开发的高通量方法来识别预处理途径，以探索轴突变性，从而可以快速筛选化合物和遗传途径。使用成人DRG神经元，我们计划筛选药物化合物和慢病毒开放式阅读框（ORF）库的库。首先，我们将优化和微型化筛选分析和图像分析（R21相），然后使用高通量筛选和成像分析来识别通过促进“预处理”响应（R33相）来增强轴突重长增长的化合物和/或基因。其次，我们将开发辅助筛选，以测定预处理，神经元亚型特异性预处理反应的分子标记，以及基于微流体的轴突生长的抑制性底物（R21期）的基于微流体的测定。这些测定将用于进一步表征主要屏幕（R33相）的“命中”。第三，我们将使用坐骨神经挤压测定法检查筛选中确定的几种优先化合物的体内活性（R33期）。通过这些实验，我们希望发现损伤引起的预处理的试剂和途径，这将导致刺激稳健的轴突再生和抑制性底物增长的新方法，这可能会导致对受损神经系统的新治疗。