Novel Factors Promoting Retinal Ganglion Cell Growth

描述(由申请人提供)：NIH路线图强调了生物活性“小分子”的发现如何有助于“分子水平上的细胞功能”的探索和新药的开发。为了反映这些分子的重要性，分子图书馆路线图的倡议之一是支持小分子文库的构建和筛选。小分子和多肽通过调节多种细胞信号通路来支持视网膜神经节细胞(RGC)的存活和轴突生长。识别更多这样的分子是这项拨款的重点。Forsklin和BDNF是最初在培养中发现的支持RGCs的分子的例子，后来在视神经损伤的动物模型中证明了它们的有效性。在细胞培养中识别支持视网膜节细胞的新型小分子有两个潜在的好处。这些分子将可能在分子水平上探索RGC功能的新途径。其次，细胞培养中支持RGC的因素是候选细胞的合理来源，这些候选细胞在保护或恢复失明视神经疾病患者(如青光眼、视神经中风和视神经炎)的视力方面可能具有翻译和治疗优势。与蛋白质等大分子相比，小分子具有潜在的优势，因为它们可能更容易被开发成药物，也可能更容易被输送到眼睛。一种合理和全面的方法来鉴定功能感兴趣的小分子是结合灵敏的生物测定和高通量筛选。我们已经成功地启动了这样的筛查，使用基于自动荧光的图像分析系统来检测免疫纯化的大鼠视网膜神经节细胞的培养，该系统测量细胞存活和轴突生长。这项建议的目的是扩大这一筛选，以便识别、验证和优化促进视网膜节细胞存活和再生的新型药物化合物。更具体地说，这笔赠款将支持继续调查RGC存活和轴突生长因子的“小分子”文库，通过证明剂量反应关系来确认这些HITS的生物活性，并进一步确定显示生物活性的基本元素。筛选中确定的试剂将成为未来假说驱动研究的主题，以确定它们在体外的作用机制，并进一步探索它们在体内的治疗潜力。

DESCRIPTION (provided by applicant): The NIH Roadmap emphasizes how the discovery of bioactive "small molecules" can help both in the exploration of "cellular functions at the molecular level" and the development of new drugs. Reflecting the importance of such molecules, one of the Molecular Libraries Roadmap's Initiatives is to support the construction and screening of small molecule libraries. Small molecules and peptides support retinal ganglion cell (RGC) survival and neurite outgrowth by modulating various cellular signaling pathways. Identification of additional such molecules is the focus of this grant. Forskolin and BDNF are examples of molecules that that were originally found to support RGCs in culture and later demonstrated to have efficacy in animal models of optic nerve injury. Identification of novel small molecules that support RGCs in cell culture offers two potential benefits. Such molecules will make possible new lines of inquiry into RGC function at the molecular level. Secondly, factors supporting RGCs in cell culture are a logical source of candidates that may have translational, therapeutic benefit in preserving or restoring vision in patients with blinding optic nerve diseases such as glaucoma, optic nerve stroke, and optic neuritis. Small molecules have potential advantages compared to larger molecules, such as proteins, in that they may be more readily developed into drugs and may be more easily delivered to the eye. A rational and comprehensive way to identify functionally interesting small molecules is to combine a sensitive bioassay with a high throughput screen. We have successfully initiated such a screen, assaying cultures of immunopurified rat RGCs with an automated fluorescence-based image analysis system that measures cell survival and neurite outgrowth. The objective of this proposal is to expand this screen so as to identify, validate, and optimize novel pharmaceutical compounds that promote survival and regeneration of RGCs. More specifically, this grant will support the continued survey of a "small molecule" library for RGC survival and neurite outgrowth factors, confirm the bioactivity of those hits by demonstrating a dose response relationship, and further determine the essential element that exhibits the bioactivity. Reagents identified in the screen will be the subject of future hypothesis driven research to characterize their mechanism of action in vitro and further explore their therapeutic potential in vivo.