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: 7373784
• 负责人: Gulgun TEZEL
• 金额: $ 37万
• 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 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7373784
• 关键词: Address; American; Animal Experiments; Axon; Biological Markers; Blindness; Cell Death; Cell Death Process; Cells; Chronic; Complement; Complex; Data Set; Detection; Discipline; Disease; Disease Progression; Down-Regulation; Eye; Gel; Glaucoma; Goals; Human; Immunohistochemistry; In Vitro; Intervention; Mass Spectrum Analysis; Modeling; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Ocular Hypertension; Phosphoproteins; Physiologic Intraocular Pressure; Post-Translational Protein Processing; Process; Proteins; Proteome; Proteomics; Rattus; Relative (related person); Retina; Retinal Ganglion Cells; Sampling; Series; Techniques; Technology; Time; Transcriptional Activation; Two-Dimensional Polyacrylamide Gel Electrophoresis; Up-Regulation; Vision; base; human disease; improved; in vivo; innovation; novel; pressure; prevent; protein expression; protein protein interaction; research study; response to injury

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（由申请人提供）：只有通过更好地了解青光眼的致病机制，才能改进和创新治疗方法。使用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睛的组织病理学研究，提供了证据表明，蛋白质表达，蛋白质相互作用，翻译后蛋白修饰和蛋白水解裂解的改变改变视网膜神经节细胞（RGC）的蛋白质补体在青光眼性神经变性的致病机制具有重要意义。本申请中提出的实验基于以下假设：使用蛋白质组学技术的创新分析方法可以大规模地鉴定RGC蛋白补体中的时间依赖性改变，其表征了RGC响应于从初始损伤到青光眼中细胞死亡的执行的损伤的精确机制。具体的目的，将解决这一假设包括在RGC蛋白表达和翻译后蛋白修饰的过程中的青光眼神经变性的慢性压力诱导的青光眼的实验大鼠模型的变化的识别。RGC蛋白质组的时间依赖性改变，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之间的蛋白质组数据集来定量评价，所述蛋白质组数据集使用通过从与眼内压（IOP）暴露和轴突损失匹配的大鼠眼合并在不同时间点获得的RGC蛋白质样品。将统计学确定差异蛋白表达与IOP暴露水平和轴突损失之间的关系。为了提高蛋白质表达的检测、鉴定和相对定量的灵敏度，将利用互补蛋白质组学方法，其将包括使用质谱法的基于2D PAGE和无凝胶技术。氧化修饰的RGC蛋白将通过2D oxylot分析鉴定，磷酸化的RGC蛋白及其在富集的磷蛋白复合物中的相互作用蛋白将通过靶向蛋白质组学的互补方法鉴定。平行实验还将使用免疫组织化学确定所鉴定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，还将利用从人类供体眼睛获得的青光眼和正常视网膜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（失明的主要原因）的神经保护干预提供生物标志物和新的治疗靶点。从这些研究中获得的信息也有望在多个学科中用于表征导致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，一种特定类型的神经细胞，视网膜神经节细胞，逐渐死亡，导致视觉功能逐渐丧失。由于目前对这种致盲性疾病的治疗不足以防止疾病进展，因此需要开发其他治疗策略来保护青光眼眼中的这些细胞。作为实现这一目标的必要条件，该项目旨在通过动物实验中使用强大的技术来确定与青光眼中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300万患有青光眼的美国人提供新的治疗可能性。