Screening for Molecules that Promote Photoreceptor Synaptogenesis

项目总结 NEI大胆的目标倡议是一项大胆的努力，旨在通过神经元再生来恢复视力 以及眼睛和视觉系统中的神经连接。哺乳动物的主要障碍之一 光感受器移植实验一直是，并将继续是低效率的整合 以及光感受器群体移植后的突触形成。为了解决这个问题 路障，并响应RFA-EY-15-002(其目标是“基于发现的方法来确定 对神经元再生至关重要的未知因素，引导它们的轴突指向靶点，并使新的 功能连接“)，我们建议将最先进的干细胞方法与高含量 筛选新的小分子和促进人类健康的分子途径 光感受器轴突生长和突触形成。为了实现这一雄心勃勃的目标，我们有 召集了一个多学科的研究小组，他们在人类视网膜干方面有多年的经验 细胞生物学、视网膜细胞和分子生物学、高含量筛选(HCS)分析的发展和药物 筛选、轴突引导、突触生物学和微流体。 研究小组成员已经进行了筛查，成功地识别出 促进小鼠视网膜神经节细胞(RGC)和其他视网膜神经元突起生长的分子， 这增加了人类干细胞来源的神经元培养中突触的形成。对于这个项目，我们 建议扩展这一先前的工作，并开发出健壮和可重复的体外轴突生长和 利用人多能干细胞获得的光感受器进行突触发生分析 衍生出三维视泡样结构，然后使用这些分析方法来识别和 表征生物学和临床上相关的分子。更具体地说，SA1将专注于 一种两层体外筛选的开发和实施，旨在识别影响 HPSC-PR轴突生长和/或引导；SA2将专注于筛查以识别增强 HPSC-PR突触标志物的表达；而SA3将重点发展检测以证实 培养中功能性PR突触的形成。这些目标和里程碑的成功完成将产生 第一个体外人体测试系统，旨在快速筛选和严格测试其分子 能够促进hPSC-PR连接。这一平台不仅要加快努力，实现 功能性PR替代，但也可以作为一个有价值的人类临床前模型系统。

PROJECT SUMMARY The NEI Audacious Goals Initiative is a bold effort to “to restore vision through regeneration of neurons and neural connections in the eye and visual system.” One of the major roadblocks in mammalian photoreceptor transplantation experiments has been, and continues to be, the low efficiency of integration and synapse formation following transplantation of photoreceptor populations. In order to address this roadblock, and in response to RFA-EY-15-002 (which is directed at “discovery-based approaches to identify unknown factors critical to the regeneration of neurons, guiding their axons to targets, and making new functional connections”), we propose to combine state-of-the-art stem cell methodologies with high content screening approaches to identify novel small molecules and molecular pathways that promote human photoreceptor axonal outgrowth and synapse formation. To accomplish this ambitious goal, we have assembled a multidisciplinary group of investigators who have years of experience in human retinal stem cell biology, retinal cell and molecular biology, high content screening (HCS) assay development and drug screening, axonal guidance, synaptic biology, and microfluidics. Members of the research team have already carried out screens that have successfully identified molecules that promote neurite outgrowth of murine retinal ganglion cells (RGCs) and other retinal neurons, and that increase synapse formation in cultures of human stem cell-derived neurons. For this project, we propose to extend this prior work and develop robust and reproducible in vitro neurite outgrowth and synaptogenesis assays using photoreceptors (PRs) obtained from human pluripotent stem cell (hPSCs) derived 3-dimensional optic vesicle-like structures, and then to use these assays to identify and characterize biologically and clinically relevant molecules. More specifically, SA1 will focus on the development and execution of a two-tiered in vitro screen designed to identify molecules that influence hPSC-PRs axon outgrowth and/or guidance; SA2 will focus on a screen to identify molecules that enhance hPSC-PR synaptic marker expression; and SA3 will focus on the development of assays to confirm functional PR synapse formation in culture. Successful completion of these aims and milestones will yield the first in vitro human assay system designed to rapidly screen and rigorously test molecules for their ability to promote hPSC-PR connectivity. This platform should not only accelerate efforts to achieve functional PR replacement in patients, but could also serve as a valuable human preclinical model system.