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

项目总结/摘要 青光眼引起的视力丧失和失明，无论起始事件如何， 最终是视网膜神经节细胞（RGC）功能障碍和死亡的结果。的一种方法 保护青光眼的视力是找到保护视网膜神经节细胞并促进其生长的方法。 健康和生存。作为威尔默眼科研究所高级研究员， 青光眼专业培训计划，我已经开始与唐纳德扎克博士在RGC工作 生物学他的实验室，通过高含量筛选方法，确定舒尼替尼作为一种有效的 RGC启动子在体外和体内存活，在应激如轴突损伤和 谷氨酸兴奋毒性舒尼替尼是一种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 受体信号传导并促进癌细胞凋亡，其对RGCs的神经保护活性 有点令人惊讶了解舒尼替尼的分子机制可以提供 重要的见解的途径介导RGC的生存，但破译其机制 因为舒尼替尼对许多不同的激酶具有活性。我们假设 一些激酶亚类参与RGC死亡，并且它们可以用RNA鉴定 干扰（RNAi）筛选。为此，我们已经调整RNAi用于RGC， 体外和体内。在特异性目标1中，我们将使用短发夹RNA（short-hairpin RNA，shRNA）敲除 候选激酶在阵列培养的鼠RGC中与基于荧光的成像结合， 鉴定提供存活优势的shRNA。在具体目标2中，我们提出了一种体内 筛选靶向鼠激酶组的合并的病毒递送的shRNA，以鉴定这些激酶 介导视神经切断引起的RGC死亡。 我已经决定积极地追求作为一名大学临床科学家的职业生涯， 提供病人护理和教育居民，但我的大部分努力都是针对 负责RGC神经保护的实验室研究项目发展 必要的技能和知识进行竞争性研究，我参加了一个 多年的指导研究项目在这里威尔默与教师，有专业知识，在高- 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病变的模型。

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.