TNF-alpha in Cell Death & Neuroprotection in 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，才能改进治疗方法。在最初拨款期间进行的研究支持肿瘤坏死因子- α (TNF-a)信号在青光眼中的参与，并阐明了青光眼神经变性期间TNF-a介导的视网膜神经节细胞（RGCs）死亡的许多方面。在这个更新申请中提出的实验是基于一个假设，即使用蛋白质组学技术的创新分析方法可以通过识别蛋白质补体中的时间依赖性改变来进一步阐明青光眼中TNF-a信号传导。解决这一假设的具体目的是：(1)在体外比较鉴定暴露于TNF-a的RGCs和胶质细胞的蛋白质组学改变；(2)在体内鉴定青光眼神经变性过程中RGCs中与TNF-a信号相关的蛋白质组学改变。在体外实验中，将从成人组织中分离RGCs和胶质细胞。为了大规模鉴定tnf -a诱导的蛋白质补体变化，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。通过比较在存在和不存在TNF-a的情况下从rgc和胶质细胞获得的蛋白质组学数据集，将定量评估蛋白质表达的时间依赖性改变。互补的方法将被用来提高蛋白质鉴定的敏感性。此外，磷酸化蛋白将通过靶向蛋白质组学使用串联质谱鉴定。所提出的比较分析的结果应在蛋白质水平上提供关于RGCs和胶质细胞对TNF-a的差异反应的全面信息。更好地了解与胶质细胞相对保护青光眼损伤相关的细胞机制，有助于类似地提高青光眼RGC的存活率。在体内实验将采用实验性青光眼大鼠模型，通过向角膜缘静脉注射高渗盐水单侧诱导眼内压（IOP）升高。在长达12周的随访期间，测定每只大鼠在不同时间点的IOP暴露和轴突损失。RGC蛋白样本将从IOP暴露和轴突损失匹配的大鼠眼睛中收集。在富集的多蛋白复合物中相互作用和磷酸化的RGC蛋白将使用靶向蛋白质组学方法进行鉴定。研究的信号复合体将包括TNF-a/TNF受体复合体以及与丝裂原活化蛋白激酶和核因子- κ b通路相关的复合体（涉及TNF-a信号转导和青光眼神经退行性变）。由于TNF-a具有多种生物活性，可促进细胞死亡和存活信号，特异性抑制细胞死亡信号和/或扩增存活信号（而不是抑制受体结合）可实现对TNF-a介导的RGC死亡的神经保护。因此，在蛋白质组范围内提高对青光眼神经退行性变过程中TNF-a信号的理解，将为青光眼（主要致盲原因）的有效神经保护干预提供新的特异性治疗靶点。

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.