High-content high-throughput functional genomics in retinal ganglion cells

项目摘要/摘要 青光眼引起的视力丧失和失明，无论是什么引发的事件，都是 最终导致视网膜神经节细胞(RGC)功能障碍和死亡。一种方法是 在青光眼中保持视力是寻找保护视网膜神经节细胞和促进其功能的方法 健康和生存。作为威尔默眼科研究所的高级研究员 青光眼专科培训计划，我已经开始与Donald Zack博士合作进行RGC 生物学。他的实验室通过高含量筛选方法，确定了舒尼替尼是一种有效的 促进RGC存活的体外和体内，在应激，如轴突损伤和 谷氨酸兴奋性毒性。孙尼替尼是FDA批准的一种具有广泛选择性的蛋白激酶抑制剂 被批准用于治疗多种癌症。鉴于舒尼替尼阻断神经营养因子 受体信号转导促进肿瘤细胞凋亡及其对视网膜节细胞的神经保护作用 有点令人惊讶。了解舒尼替尼的分子机制可以提供 对调节RGC存活的途径的重要见解，但破译其机制 一直具有挑战性，因为舒尼替尼对许多不同的激酶都有活性。我们假设 RGC死亡过程中涉及一些激酶的子集，并且可以使用RNA来识别它们 基于干扰(RNAi)的屏幕。为此，我们已将RNAi修改为在RGC中使用 体外和体内。在特定的目标1中，我们将使用短发夹状RNA(ShRNA)来敲除 阵列化培养的小鼠视网膜神经节细胞中的候选激酶与基于荧光的成像相结合 确定提供生存优势的shRNA。在具体目标2中，我们提出了一种体内 筛选以小鼠基因组为靶点的汇集的、病毒传递的shRNA以识别这些激酶 调节视神经切断后RGC的死亡。 我已经决定积极地追求大学临床科学家的职业生涯。 提供病人护理和教育住院医生，但我的大部分努力都是针对 负责监督RGC神经保护方面的积极实验室研究项目。发展，发展 进行竞争性研究所需的技能和知识，我正在参加一个 威尔默大学的多年指导研究项目，其教职员工拥有高- 内容、高通量筛选、RGC信号转导和神经保护以及动物 青光眼和其他视神经疾病的模型。

Project Summary/Abstract Vision loss and blindness from glaucoma, regardless of the initiating event, are ultimately the result of dysfunction and death of retinal ganglion cells (RGCs). One approach to preserving vision in glaucoma is to find ways to protect retinal ganglion cells and promote their health and survival. As a current fellow in the Wilmer Ophthalmological Institute's Advanced Specialty Training Program in Glaucoma, I have begun working with Dr. Donald Zack on RGC biology. His lab, through a high content screening approach, identified sunitinib as a potent promoter of RGC survival both in vitro and in vivo, following stresses such as axonal injury and glutamate excitotoxicity. Sunitinib is a broadly selective protein kinase inhibitor that is FDA approved for the treatment of a variety of cancers. Given that sunitinib blocks neurotrophin receptor signaling and promotes apoptosis in cancer cells, its neuroprotective activity on RGCs is somewhat surprising. Understanding sunitinib's molecular mechanism could provide important insights into the pathways mediating RGC survival, but deciphering its mechanism has been challenging because sunitinib is active against many different kinases. We hypothesize that some subset of kinases are involved in RGC death and that they can be identified using RNA interference (RNAi)-based screens. To this end, we have adapted RNAi for use in RGCs both in vitro and in vivo. In Specific Aim 1, we will use short-hairpin RNA (shRNA) to knockdown candidate kinases in arrayed cultured murine RGCs coupled with fluorescence-based imaging to identify shRNAs that provide a survival advantage. In Specific Aim 2, we propose an in vivo screen of pooled, virally delivered shRNAs targeting the murine kinome to identify those kinases that mediate RGC death in response to optic nerve transection. I have decided to pursue a career as a university-based clinician scientist, actively providing patient care and educating residents, but with a majority of my effort being directed towards overseeing an active laboratory research program in RGC neuroprotection. To develop the necessary skills and knowledge to conduct competitive research, I am participating in a multi-year mentored research project here at Wilmer with faculty that have expertise in high- content, high-throughput screening, RGC signal transduction and neuroprotection, and animal models of glaucoma and other optic neuropathies.