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

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：
8780632
负责人：
Derek Stuart Welsbie
金额：
$ 23.19万
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 2015-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8780632
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 High-Throughput Nucleotide Sequencing Image In Vitro Injury Institutes Knowledge Laboratory Research Libraries Malignant Neoplasms Mediating Mentors Modeling Molecular Mus Nerve Growth Factor Receptors Neurons Optic Nerve Injuries Optic Nerve Transections Pathway interactions Patient Care Phosphotransferases Pigmentary Glaucoma Plasmids Protein Kinase Inhibitors Protocols documentation RNA Interference Receptor Signaling Research Research Project Grants Retinal Ganglion Cells 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 axon injury base cancer cell career excitotoxicity functional genomics ganglion cell gene therapy genome-wide analysis 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.

项目概要/摘要无论起始事件如何，青光眼导致的视力丧失和失明都是最终是视网膜神经节细胞（RGC）功能障碍和死亡的结果。一种方法保护青光眼患者的视力就是找到保护视网膜神经节细胞并促进其恢复的方法健康和生存。作为威尔默眼科研究所高级研究员青光眼专业培训项目，我已开始与 Donald Zack 博士一起开展 RGC 工作生物学。他的实验室通过高内涵筛选方法，确定舒尼替尼是一种有效的药物在轴突损伤和应激等应激后，RGC 在体外和体内存活的促进剂谷氨酸兴奋毒性。舒尼替尼是一种广泛选择性蛋白激酶抑制剂，已获得 FDA 批准被批准用于治疗多种癌症。鉴于舒尼替尼会阻断神经营养素受体信号传导并促进癌细胞凋亡及其对 RGC 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对介导 RGC 存活的途径有重要见解，但破译其机制由于舒尼替尼对许多不同的激酶具有活性，因此一直具有挑战性。我们假设某些激酶子集与 RGC 死亡有关，并且可以使用 RNA 来识别它们基于干扰（RNAi）的筛选。为此，我们对 RNAi 进行了改造，用于 RGC 中体外和体内。在具体目标 1 中，我们将使用短发夹 RNA (shRNA) 进行基因敲除阵列培养的鼠 RGC 中的候选激酶与基于荧光的成像相结合识别提供生存优势的 shRNA。在具体目标 2 中，我们提出了一种体内筛选针对鼠激酶组的汇集的、病毒传递的 shRNA，以识别这些激酶介导 RGC 因视神经横断而死亡。我决定积极从事大学临床科学家的职业提供病人护理和教育居民，但我的大部分努力都是针对监督 RGC 神经保护方面活跃的实验室研究项目。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，教职人员拥有高水平的专业知识内容、高通量筛选、RGC信号转导和神经保护、动物青光眼和其他视神经病变的模型。