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。在最初的资助期间正在进行的研究支持了青光眼中肿瘤坏死因子-α（TNF-α）信号传导的参与，并阐明了青光眼神经变性期间TNF-α介导的视网膜神经节细胞（RGC）死亡的许多方面。在该更新申请中提出的实验基于以下假设：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中的时间依赖性改变来进一步阐明青光眼中的TNF-α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-α的RGCs和神经胶质中的蛋白质组学改变;和（2）在体内鉴定在肿瘤性神经变性过程中与RGCs中TNF-α信号传导相关的蛋白质组学改变。对于体外实验，将从成体组织中分离RGC和神经胶质细胞。为了大规模鉴定TNF-α诱导的蛋白质补体的改变，差异蛋白质组学将利用从培养的细胞获得的蛋白质裂解物。通过比较从在存在和不存在TNF-α的情况下孵育的RGC和神经胶质细胞获得的蛋白质组数据集，定量评价蛋白质表达的时间依赖性变化。将利用补充方法来提高蛋白质鉴定的灵敏度。此外，磷酸化蛋白质将通过靶向蛋白质组学使用串联质谱法进行鉴定。拟议的比较分析的结果应提供全面的信息，RGCs和神经胶质细胞的差异反应，TNF-α在蛋白质水平。更好地理解与胶质细胞对青光眼损伤的相对保护相关的细胞机制可以促进类似地改善青光眼中RGC存活的努力。拟议的体内实验将利用青光眼实验大鼠模型，其中通过向角膜缘静脉注射高渗盐水来单方面诱导眼内压（IOP）升高。在长达12周的随访期间，将测定在不同时间点处死的每只大鼠的IOP暴露和轴突损失。将从与IOP暴露和轴突损失匹配的大鼠眼睛中合并RGC蛋白样品。将使用靶向蛋白质组学方法鉴定富集的多蛋白复合物中的相互作用和磷酸化RGC蛋白。所研究的信号复合物将包括TNF-α/TNF受体复合物和与促分裂原活化蛋白激酶和核因子-κ B通路（其参与TNF-α信号传导和神经退行性疾病）相关的那些复合物。由于促进细胞死亡和存活信号的TNF-α的不同生物活性，细胞死亡信号传导的特异性抑制和/或存活信号传导的扩增（而不是受体结合的抑制）应实现针对TNF-α介导的RGC死亡的神经保护。因此，在蛋白质组范围内对青光眼神经变性过程中TNF-α信号传导的更好理解应该为青光眼（失明的主要原因）的有效神经保护干预提供新的和特定的治疗靶点。

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.