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

描述(由申请人提供)：青光眼导致的视力丧失和失明，无论起始事件是什么，最终都是视网膜神经节细胞(RGC)功能障碍和死亡的结果。保护青光眼视力的一种方法是找到保护视网膜神经节细胞的方法，促进它们的健康和生存。作为Wilmer眼科研究所青光眼高级专科培训项目的现任研究员，我已经开始与Donald Zack博士合作研究RGC生物学。他的实验室通过一种高含量的筛选方法，在轴突损伤和谷氨酸兴奋毒性等应激反应之后，确定Sunitinib是RGC在体外和体内存活的有效促进剂。舒尼替尼是一种具有广泛选择性的蛋白激酶抑制剂，FDA批准其用于治疗各种癌症。鉴于苏尼替尼阻断神经营养素受体信号转导并促进癌细胞凋亡，其对视网膜节细胞的神经保护作用有些令人惊讶。了解苏尼替尼的分子机制可以为调节RGC存活的途径提供重要的见解，但破译其机制一直是具有挑战性的，因为苏尼替尼对许多不同的激酶具有活性。我们假设RGC死亡与某些激酶有关，并且可以使用基于RNA干扰(RNAi)的筛选来识别它们。为此，我们已经将RNAi用于体外和体内的视网膜节细胞。在具体目标1中，我们将使用短发夹状RNA(ShRNA)敲除排列培养的小鼠视网膜节细胞中的候选激酶，并结合基于荧光的成像来识别提供生存优势的shRNA。在特定的目标2中，我们提出了一种针对小鼠基因组的联合的、病毒递送的shRNAs的体内筛选，以识别那些介导视神经横断反应中RGC死亡的激酶。我决定从事大学临床科学家的职业，积极提供患者护理和教育住院医生，但我的大部分时间都被用于监督RGC神经保护方面的积极实验室研究项目。为了培养进行竞争性研究所需的技能和知识，我在Wilmer参加了一个为期多年的导师研究项目，该项目的教职员工拥有高内容、高通量筛查、RGC信号转导和神经保护以及青光眼和其他视神经疾病的动物模型方面的专业知识。 与公共健康相关：青光眼导致的视力丧失是由视网膜神经节细胞死亡引起的，视网膜神经节细胞是将视力从眼睛输送到大脑的重要神经细胞。我们正在使用基因疗法，试图更好地理解触发这些神经节细胞死亡的信号。通过这样做，我们希望找到新的药物靶点，可以用来防止细胞死亡和视力丧失，并提供青光眼的新治疗方法。

DESCRIPTION (provided by applicant): Vision loss and blindness from glaucoma, regardless of the initiating event, are ultimately the result of dysfunction and death of retinal ganglion cels (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 effor 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. PUBLIC HEALTH RELEVANCE: Vision loss from glaucoma is caused by the death of retinal ganglion cells, important nerve cells that carry vision from the eye to the brain. We are using gene therapy to try to better understand the signals that trigger these ganglion cells to die. In doing so, we hope to identify new drug targets that could be used to prevent cell death and vision loss and offer new treatments for glaucoma.