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发病机制，才能改进治疗方法。在最初的资助期间正在进行的研究已经支持了肿瘤坏死因子-α信号在青光眼中的参与，并阐明了在青光眼神经变性过程中肿瘤坏死因子-α介导的视网膜神经节细胞(RGCs)死亡的许多方面。在这一更新应用中提出的实验是基于这样一个假设，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与时间相关的变化来进一步阐明青光眼中的肿瘤坏死因子-a信号。解决这一假说的具体目标是：(1)在体外比较鉴定暴露于肿瘤坏死因子-a的视网膜节细胞和神经胶质细胞的蛋白质组改变；(2)在体内确定青光眼神经变性过程中视网膜节细胞中与肿瘤坏死因子-α信号相关的蛋白质组改变。对于体外实验，视网膜神经节细胞和神经胶质细胞将从成人组织中分离出来。为了大规模鉴定肿瘤坏死因子-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获得的蛋白质裂解产物。蛋白质表达随时间的变化将通过比较视网膜神经节细胞和胶质细胞在存在和不存在肿瘤坏死因子-α的情况下孵育的蛋白质组数据集进行定量评估。将利用互补的方法来提高蛋白质鉴定的灵敏度。此外，还将使用串联质谱仪通过靶向蛋白质组学鉴定磷酸化蛋白质。拟议的比较分析结果应该在蛋白质水平上提供有关视网膜节细胞和神经胶质细胞对肿瘤坏死因子-a的差异反应的全面信息。更好地了解与相对保护胶质细胞免受青光眼损伤相关的细胞机制，可以促进类似地改善青光眼RGC存活率的努力。拟议的体内实验将利用青光眼的实验大鼠模型，在该模型中，向角膜缘静脉注射高渗盐水将单侧诱导眼压(IOP)升高。在长达12周的随访期内，将在不同的时间点确定每只大鼠的眼压暴露和轴突丢失。RGC蛋白样本将从符合眼压暴露和轴突丢失的大鼠眼睛中提取。在浓缩的多蛋白复合体中相互作用和磷酸化的RGC蛋白将使用有针对性的蛋白质组学方法进行鉴定。所研究的信号复合体将包括肿瘤坏死因子-a/肿瘤坏死因子受体复合体，以及与丝裂原活化蛋白激酶和核因子-kappaB通路(参与肿瘤坏死因子-a信号和青光眼神经变性)相关的信号复合体。由于肿瘤坏死因子-α具有促进细胞死亡和存活信号的多种生物活性，特异性抑制细胞死亡信号和/或生存信号的放大(而不是受体结合的抑制)，应该对肿瘤坏死因子-α介导的RGC死亡起到神经保护作用。因此，在蛋白质组范围内对青光眼神经变性过程中肿瘤坏死因子-α信号的更好理解应该为青光眼的有效神经保护干预提供新的和特定的治疗靶点，青光眼是导致失明的主要原因。

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.