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

基本信息

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
项目类别：
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

项目摘要

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）：仅通过更好地理解青光眼的致病机制，才能改善和创新的治疗方法。使用不同系列体外和体内实验的初步研究，以及使用人眼的组织病理学研究，证明了蛋白质表达的改变，蛋白质蛋白质相互作用，翻译后蛋白质修饰和蛋白水解裂解改变了蛋白质补充的蛋白质补充在青光眼神经变性过程中，神经节细胞（RGC）在致病机制中具有重要意义。本应用程序中提出的实验是基于以下假设：使用蛋白质组学技术的创新分析方法可以识别RGC蛋白质中的时间依赖性变化，这表征了RGC对从最初损害到执行的损伤的精确机制，这表征了RGC响应的精确机制。青光眼中细胞死亡的死亡。将解决该假设的具体目的包括在慢性压力诱导的青光眼的实验大鼠模型中鉴定RGC蛋白表达的变化和在青光眼神经变性过程中翻译蛋白修饰的变化。 RGC蛋白质组的时间依赖性变化，例如提高调节或蛋白质表达的调节，将通过使用在不同的时间点获得的RGC蛋白样品在眼睛高血压和对照眼中进行比较，从而定量评估蛋白质组学的数据集，并通过从大鼠眼中汇集的不同时间点获得的RGC蛋白样品来评估。眼内压（IOP）暴露和轴突损失。差异蛋白表达与IOP暴露水平与轴突损失之间的关系将在统计上确定。为了提高蛋白质表达的检测，鉴定和相对定量的敏感性，将利用互补的蛋白质组学方法，其中将包括基于2D页面的基于2D Page和gel无凝胶技术。氧化修饰的RGC蛋白将通过2D Oxylot分析鉴定，并且将通过靶向蛋白质组学的互补方法来鉴定磷酸化的RGC蛋白及其相互作用的蛋白质。平行实验还将确定使用免疫组织化学的细胞定位。除了从眼部高血压和对照大鼠眼睛获得的组织学切片外，还将利用从人类供体眼中获得的青光眼和正常视网膜，以更好地验证新发现与人类疾病的相关性。这些研究应提供有关蛋白质水平上与RGC死亡相关的细胞机制的全面信息，从而为青光眼中神经保护干预措施提供生物标志物和新型治疗靶标，这是盲目失明的主要原因。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，一种特定类型的神经细胞中，视网膜神经节细胞逐渐死亡，导致视觉功能的逐渐丧失。由于目前对这种盲人疾病的治疗不足以防止疾病进展，因此需要制定其他治疗策略来保护这些细胞在青光眼眼中。为了实现这一目标，该项目旨在使用动物实验中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，并利用人类捐助者的眼睛进行实验。预计这些研究将提供有关神经细胞死亡过程的全面信息，从而为300万美国人患有青光眼的美国人提供新的治疗可能性。