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

啮齿动物视网膜神经节细胞的高内涵高通量功能基因组学

基本信息

批准号：
8735206
负责人：
Derek Stuart Welsbie
金额：
$ 3.17万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-12-01 至 2016-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8735206
关键词：
Animal Model Apoptosis Axon Axotomy Blindness Brain Cause of Death Cell Death Cell Survival Cells Cellular biology Cessation of life Coupled Dose Drug Targeting Electroporation Event Eye FDA approved Faculty Fluorescence Frequencies Functional disorder Genetic Genome Glaucoma Glutamates Harvest Health Image In Vitro Injury Institutes Knowledge Laboratory Research Libraries Malignant Neoplasms Mediating Mentors Modeling Molecular Mus Nerve Growth Factor Receptors Neurons Open-Angle Glaucoma Optic Nerve Injuries Optic Nerve Transections Pathway interactions Patient Care Phosphotransferases Plasmids Protein Kinase Inhibitors Protocols documentation RNA Interference Receptor Signaling Research Research Project Grants Retinal Ganglion Cells Rodent Scientist Signal Pathway Signal Transduction Site Sorting - Cell Movement Specificity Stress Subfamily lentivirinae System Time Training Programs Tyrosine Kinase Inhibitor Universities Viral Vision Work abstracting base cancer cell career excitotoxicity functional genomics ganglion cell gene therapy genome wide association study high throughput screening in vivo in vivo Model inhibitor/antagonist injured insight knock-down loss of function medical specialties neuroprotection optic nerve disorder prevent programs promoter protective effect protein kinase inhibitor receptor research study response screening skills small hairpin RNA small molecule

项目摘要

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.

项目摘要/摘要不管发生事件如何，青光眼的视力丧失和失明是最终，视网膜神经节细胞（RGC）功能障碍和死亡的结果。一种方法保持青光眼的视力是找到保护视网膜神经节细胞并促进其促进其的方法健康和生存。作为威尔默眼科研究所的现任研究员青光眼专业培训计划，我已经开始与Donald Zack博士一起在RGC上合作生物学。他的实验室通过高内容筛选方法将舒尼尼确定为有效 RGC生存的启动子在体外和体内都有符合轴突损伤和等应力谷氨酸兴奋性。 Sunitinib是一种广泛选择性蛋白激酶抑制剂，是FDA 批准用于治疗各种癌症。鉴于圣替尼阻止了神经营养蛋白受体信号传导并促进癌细胞中的凋亡，其神经保护活性在RGC上有些令人惊讶。了解舒尼替尼的分子机制可以提供对介导RGC生存的途径的重要见解，但破译了其机制之所以具有挑战性，是因为Sunitinib对许多不同的激酶有活跃。我们假设一部分激酶参与RGC死亡，可以使用RNA识别它们干扰（RNAi）基于屏幕。为此，我们改编了RNAi用于RGCS 体内和体内。在特定的目标1中，我们将使用短发RNA（shRNA）敲击阵列培养的鼠RGC中的候选激酶以及基于荧光成像的候选激酶确定提供生存优势的shRNA。在特定目标2中，我们提出一个体内靶向鼠类活色组的合并，病毒传递的shRNA的屏幕以识别那些激酶该响应视神经横向介导RGC死亡。我决定从事大学临床医生的职业，积极地提供患者护理和教育居民，但我的大部分努力都被指导旨在监督RGC神经保护方面的活跃实验室研究计划。发展进行竞争研究的必要技能和知识，我正在参加威尔默的多年指导研究项目，教师在高中有专业知识内容，高通量筛查，RGC信号转导和神经保护以及动物青光眼和其他视力神经病的模型。