Proteomic Analysis of Retinal Ganglion Cell Death in Glaucoma

青光眼视网膜神经节细胞死亡的蛋白质组学分析

• 批准号: 7741650
• 负责人: Gulgun TEZEL
• 金额: $ 36.63万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-12-01 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7741650
• 关键词: Address; Affect; American; Animal Experiments; Axon; Axoplasmic Streaming; Biological Markers; Blindness; Cell Death; Cell Death Process; Cell physiology; Cells; Chronic; Complement; Complex; Data; Data Set; Databases; Detection; Development; Discipline; Disease; Disease Progression; Distal; Down-Regulation; Ensure; Event; Experimental Designs; Experimental Models; Eye; Foundations; Funding; Future; Gel; Glaucoma; Goals; Health; Human; Immunohistochemistry; In Vitro; Injury; Interruption; Intervention; Investigation; Mass Spectrum Analysis; Modeling; Modification; Nerve Degeneration; Nerve Tissue Proteins; Neurodegenerative Disorders; Neuroglia; Neurons; Ocular Hypertension; Optic Disk; Optic Nerve; Phosphoproteins; Phosphorylation; Physiologic Intraocular Pressure; Population; Post-Translational Protein Processing; Procedures; Process; Proteins; Proteome; Proteomics; Published Comment; Rattus; Reactive Oxygen Species; Relative (related person); Research; Research Personnel; Retina; Retinal Ganglion Cells; Sampling; Series; Signal Transduction; Suggestion; Techniques; Technology; Time; Tissues; Trust; Two-Dimensional Polyacrylamide Gel Electrophoresis; Up-Regulation; Vision; Work; base; experience; human disease; improved; in vivo; injured; innovation; innovative technologies; neuronal cell body; novel; oxidative damage; pressure; prevent; protein expression; protein protein interaction; research study; response; response to injury; success; treatment strategy

DESCRIPTION (provided by applicant): Only through a better understanding of the pathogenic mechanisms of glaucoma will improved and innovative treatments evolve. Preliminary studies using different series of in vitro and in vivo experiments, as well as histopathological studies using human donor eyes, have provided evidence that alterations in protein expression, protein protein interactions, post translational protein modifications, and proteolytic cleavage change the protein complement of retinal ganglion cells (RGCs) during glaucomatous neurodegeneration with important implications in pathogenic mechanisms. The proposed experiments in this application are based on the hypothesis that an innovative analytical approach using the proteomics technology can identify time dependent alterations in the RGC protein complement on a large scale, which characterize precise mechanisms of the RGC response to injury from initial insult to execution of cell death in glaucoma. The specific aims that will address this hypothesis include the identification of alterations in the RGC protein expression and post translational protein modifications during the course of glaucomatous neurodegeneration in an experimental rat model of chronic pressure induced glaucoma. Time dependent alterations of the RGC proteome, such as up regulation or down regulation of protein expression, will be quantitatively evaluated by comparing the proteomic datasets between ocular hypertensive and control eyes using RGC protein samples obtained at different time points by pooling from rat eyes matched for intraocular pressure (IOP) exposure and axon loss. Relationship between the differential protein expression and the level of IOP exposure and axon loss will be statistically determined. To improve the sensitivity of the detection, identification, and relative quantification of protein expression, complementary proteomic approaches will be utilized, which will include 2D PAGE based and gel free techniques using mass spectrometry. Oxidatively modified RGC proteins will be identified through 2D oxylot analysis, and phosphorylated RGC proteins and their interacting proteins in enriched phosphoprotein complexes will be identified through complementary approaches of the targeted proteomics. Parallel experiments will also determine cellular localization of the identified proteins using immunohistochemistry. In addition to histological sections obtained from ocular hypertensive and control rat eyes, glaucomatous and normal retinas obtained from human donor eyes will also be utilized to better validate the relevance of new findings to human disease. These studies should provide comprehensive information about the cellular mechanisms associated with RGC death at the protein level, thereby offering biomarkers and novel treatment targets for neuroprotective interventions in glaucoma, a leading cause of blindness. The information obtained from these studies is also expected to be useful in multiple disciplines to characterize pathogenic processes of various other neurodegenerative diseases leading to RGC death. In glaucoma, a specific type of nerve cells, the retinal ganglion cells, progressively die leading to gradual loss of visual function. Since the current treatment of this blinding disease is not sufficient to prevent disease progression, additional treatment strategies need to be developed to protect these cells in glaucomatous eyes. As a requirement to accomplish this goal, this project aims to identify the precise mechanisms associated with the nerve cell death in glaucoma using a powerful technology in animal experiments, as well as performing experiments utilizing human donor eyes. These studies are expected to provide comprehensive information about the nerve cell death process, thereby allowing new treatment possibilities for 3 million Americans suffering from glaucoma.

描述(申请人提供)：只有通过更好地了解青光眼的发病机制，才能改进和发展创新的治疗方法。通过不同系列的体外和体内实验的初步研究，以及使用人类供体眼的组织病理学研究，已有证据表明，在青光眼神经变性过程中，蛋白质表达、蛋白质相互作用、翻译后蛋白质修饰和蛋白质水解性切割的变化改变了视网膜神经节细胞(RGCs)的蛋白质补体，在发病机制中具有重要意义。本应用中提出的实验是基于这样一个假设，即使用蛋白质组学技术的创新分析方法可以大规模识别RGC蛋白补体的随时间变化，这表征了青光眼中RGC对从最初的侮辱到细胞死亡的损伤反应的精确机制。解决这一假说的具体目标包括鉴定慢性压迫性青光眼大鼠模型青光眼神经变性过程中RGC蛋白表达的变化和翻译后蛋白修饰。RGC蛋白质组随时间的变化，例如蛋白质表达的上调或下调，将通过比较高眼压和对照眼的蛋白质组数据集，使用不同时间点从与眼压暴露和轴突丢失匹配的大鼠眼睛获得的RGC蛋白质样本来定量评估。差异蛋白表达与眼压暴露水平和轴突丢失之间的关系将在统计学上确定。为了提高蛋白质表达的检测、鉴定和相对定量的灵敏度，将使用互补的蛋白质组学方法，包括基于2D PAGE的蛋白质组学方法和使用质谱学的无凝胶技术。氧化修饰的RGC蛋白将通过2D Oxlot分析来鉴定，而磷酸化的RGC蛋白及其相互作用蛋白将通过靶向蛋白质组学的互补方法来鉴定。平行实验还将利用免疫组织化学方法确定识别出的蛋白质的细胞定位。除了从高眼压和对照大鼠眼睛获得的组织切片外，从人类供体眼睛获得的青光眼和正常视网膜也将被用来更好地验证新发现与人类疾病的相关性。这些研究应该在蛋白质水平上提供与RGC死亡相关的细胞机制的全面信息，从而为青光眼的神经保护干预提供生物标志物和新的治疗靶点。青光眼是导致失明的主要原因。从这些研究中获得的信息也有望在多个学科中有用，以表征导致RGC死亡的各种其他神经退行性疾病的致病过程。 在青光眼中，一种特殊类型的神经细胞，即视网膜神经节细胞，会逐渐死亡，导致视觉功能逐渐丧失。由于目前对这种致盲疾病的治疗不足以防止疾病的进展，需要开发更多的治疗策略来保护青光眼患者的这些细胞。作为实现这一目标的要求，该项目旨在利用动物实验中的强大技术来确定与青光眼神经细胞死亡相关的精确机制，以及利用人类供体眼睛进行实验。这些研究有望提供有关神经细胞死亡过程的全面信息，从而为300万美国青光眼患者提供新的治疗可能性。