TNF-alpha in Cell Death & Neuroprotection in Glaucoma

TNF-α 与细胞死亡

• 批准号: 7882346
• 负责人: Gulgun TEZEL
• 金额: $ 32.97万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7882346
• 关键词: Address; Adult; Axon; Blindness; Cell Death; Cell Death Signaling Process; Cell Survival; Cells; Cessation of life; Complement; Complex; Cultured Cells; Data Set; Eye; Glaucoma; Grant; Immunohistochemistry; In Vitro; Incubated; Injection of therapeutic agent; Injury; Intervention; Mediating; Microglia; Mitogen-Activated Protein Kinases; Modeling; Nerve Degeneration; Neuroglia; Nuclear; Ocular Hypertension; Pathway interactions; Physiologic Intraocular Pressure; Predisposition; Proteins; Proteome; Proteomics; Rattus; Relative (related person); Research Personnel; Retina; Retinal; Retinal Ganglion Cells; Saline; Sampling; Signal Transduction; Technology; Time; Tissues; Tumor Necrosis Factor Receptor; Tumor Necrosis Factor-alpha; Veins; base; comparative; follow-up; human TNF protein; improved; in vivo; innovation; limbal; macroglia; neuronal survival; neuroprotection; prevent; programs; protein complex; protein expression; receptor binding; research study; response; tandem mass spectrometry

DESCRIPTION (provided by applicant): Only through a better understanding of the pathogenic mechanisms of glaucoma will improved treatments evolve. Ongoing studies during the original grant period have supported the involvement of tumor necrosis factor-alpha (TNF-a) signaling in glaucoma and illuminated many aspects of the TNF-a-mediated death of retinal ganglion cells (RGCs) during glaucomatous neurodegeneration. The proposed experiments in this renewal application are based on the hypothesis that an innovative analytical approach using proteomics technology can further elucidate TNF-a signaling in glaucoma by identifying time-dependent alterations in the protein complement. The specific aims that will address this hypothesis are: (1) To comparatively identify proteomic alterations in RGCs and glia exposed to TNF-a, in vitro; and (2) To identify proteomic alterations associated with TNF-a signaling in RGCs during the course of glaucomatous neurodegeneration, in vivo. For in vitro experiments, RGCs and glial cells will be isolated from adult tissues. For large-scale identification of TNF-a-induced alterations in the protein complement, differential proteomics will utilize protein lysates obtained from cultured cells. Time-dependent alterations in protein expression will be quantitatively evaluated by comparing the proteomic datasets obtained from RGCs and glial cells incubated in the presence and absence of TNF-a. Complementary approaches will be utilized to increase the sensitivity of protein identification. In addition, phosphorylated proteins will be identified through targeted proteomics using tandem mass spectrometry. Findings of the proposed comparative analyses should provide comprehensive information about differential responses of RGCs and glia to TNF-a at the protein level. A better understanding of the cellular mechanisms associated with the relative protection of glial cells against glaucomatous injury can facilitate efforts to similarly improve RGC survival in glaucoma. The proposed in vivo experiments will utilize an experimental rat model of glaucoma in which intraocular pressure (IOP) elevation will be unilaterally induced by hypertonic saline injections into limbal veins. IOP exposure and axon loss will be determined for each rat sacrificed at different time points during a follow-up period of up to 12 weeks. RGC protein samples will be pooled from rat eyes matched for IOP exposure and axon loss. Interacting and phosphorylated RGC proteins in enriched multi-protein complexes will be identified using targeted proteomic approaches. The signaling complexes studied will include the TNF-a/TNF receptor complex and those associated with mitogen-activated protein kinase and nuclear factor-kappaB pathways (which are involved in TNF-a signaling and glaucomatous neurodegeneration). Due to diverse bioactivities of TNF-a, which promote both cell death and survival signals, specific inhibition of cell death signaling and/or the amplification of survival signaling (rather than the inhibition of receptor binding), should accomplish neuroprotection against TNF-a-mediated RGC death. An improved understanding of TNF-a signaling during glaucomatous neurodegeneration in a proteome-wide scale should therefore provide new and specific treatment targets for effective neuroprotective interventions in glaucoma, a leading cause of blindness.

描述（由申请人提供）：仅通过更好地理解青光眼的致病机制，才能改善治疗方法。原始赠款期间正在进行的研究支持青光眼中肿瘤坏死因子-Alpha（TNF-A）信号的参与，并照亮了gla糖神经变性过程中视网膜神经节细胞（RGC）的TNF-A介导的死亡的许多方面。本更新应用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通过鉴定蛋白质补体的时间依赖性改变来进一步阐明青光眼中的TNF-A信号传导。解决这一假设的具体目的是：（1）在体外暴露于TNF-A的RGC和Glia中相对识别蛋白质组学改变； （2）在体内识别RGC中与RGC中TNF-A信号传导相关的蛋白质组学改变，体内。对于体外实验，将从成人组织中分离出RGC和神经胶质细胞。为了大规模鉴定TNF-A诱导的蛋白质补体改变，差异蛋白质组学将利用从培养细胞中获得的蛋白质裂解物。时间依赖性的蛋白质表达改变将通过比较从TNF-A存在和不存在的RGC和胶质细胞中获得的蛋白质组学数据集进行定量评估。互补方法将用于提高蛋白质鉴定的敏感性。另外，将使用串联质谱法通过靶向蛋白质组学来鉴定磷酸化的蛋白质。所提出的比较分析的发现应提供有关RGC和Glia对TNF-A在蛋白质水平上的差异反应的全面信息。更好地理解与神经胶质细胞对青光眼损伤相对保护相关的细胞机制可以促进类似地提高青光眼中RGC存活的努力。所提出的体内实验将利用青光眼的实验大鼠模型，其中眼内压（IOP）升高将由高渗盐水注射到层状静脉中单方面诱导。在最多12周的随访期间，将确定在不同时间点处置的每只大鼠的IOP暴露和轴突损失。 RGC蛋白样品将从与IOP暴露和轴突损失的大鼠眼中合并。将使用靶向蛋白质组学方法鉴定富集的多蛋白质复合物中的相互作用和磷酸化的RGC蛋白。所研究的信号传导复合物将包括TNF-A/TNF受体复合物以及与有丝分裂原激活蛋白激酶和核因子-Kappab途径相关的（与TNF-A信号传导和胶状神经变性有关）。由于TNF-A的生物活性多样化，促进细胞死亡和生存信号，对细胞死亡信号传导的特定抑制和/或生存信号的扩增（而不是受体结合的抑制），应实现针对TNF-A--A--A--A--A--A-的神经保护作用介导的RGC死亡。因此，对蛋白质组范围内的青光眼神经退行性变性期间TNF-A信号传导的了解得到了进一步的了解，应为有效的神经保护干预措施提供新的和特定的治疗靶标，这是盲目的主要原因。