Proteomic Analysis of Retinal Ganglion Cell Death in Glaucoma

描述（由申请人提供）：只有更好地了解青光眼的发病机制，才能改进和创新治疗方法。利用不同系列的体外和体内实验，以及利用人供眼进行的组织病理学研究，已经提供了证据，证明在青光眼神经变性过程中，蛋白表达、蛋白相互作用、翻译后蛋白修饰和蛋白水解裂解的改变改变了视网膜神经节细胞（RGCs）的蛋白补体，对致病机制具有重要意义。本应用中提出的实验是基于一种假设，即使用蛋白质组学技术的创新分析方法可以大规模地识别RGC蛋白补体中的时间依赖性变化，这些变化表征了青光眼中RGC对从最初的损伤到细胞死亡的损伤反应的精确机制。解决这一假设的具体目标包括在慢性压力性青光眼实验大鼠模型中青光眼神经变性过程中RGC蛋白表达和翻译后蛋白修饰的改变。RGC蛋白组的时间依赖性改变，如蛋白表达的上调或下调，将通过比较眼高血压和对照眼的蛋白质组学数据集进行定量评估，这些数据集使用的是在不同时间点从眼压（IOP）暴露和轴突损失匹配的大鼠眼睛中收集的RGC蛋白样本。差异蛋白表达与IOP暴露水平和轴突损失之间的关系将被统计确定。为了提高检测、鉴定和相对定量蛋白质表达的灵敏度，将利用互补的蛋白质组学方法，包括基于2D PAGE和使用质谱的无凝胶技术。氧化修饰的RGC蛋白将通过二维氧lot分析鉴定，磷酸化的RGC蛋白及其在富集的磷酸化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。平行实验也将确定使用免疫组织化学鉴定的蛋白质的细胞定位。除了从高血压大鼠和对照大鼠的眼睛中获得的组织学切片外，还将利用从人类供体眼睛中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。

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.