Molecular mechanisms of programmed necrosis in the ischemic retina

项目摘要 视网膜缺血再灌注损伤是世界范围内视力损害的常见原因， 患者的生活质量和功能状态。视网膜神经节细胞（RGC）的存活对视力至关重要。然而，在这方面， 这些视网膜神经元非常敏感，并且它们中的许多在IR后经历坏死和凋亡。 由于RGC凋亡是通过程序化机制执行的，并且可以被调节，因此引起了人们的极大关注。 这种类型的细胞死亡。与此同时，RGC坏死没有得到足够的 考虑，因为它被视为一个偶然的和不受管制的细胞事件。我们现在知道 坏死与凋亡一样，可以通过程序化机制来执行。这种形式的坏死细胞死亡是 叫做坏死性凋亡我们已发表的数据和文献表明，RGC坏死性凋亡有助于IR诱导的 通过直接损失RGC和诱导相关的炎症反应导致视网膜损伤。所以既然 RGC坏死性凋亡是由程序化机制执行的，可以调节，这一研究领域是 非常重要。然而，调节IR诱导的RGC坏死性凋亡的信号级联仍然存在， 未知这个项目的长期目标是确定调节RGC的信号级联 根据我们已发表的数据、初步研究和已发表的文献，我们提出了一种新的治疗方法， IR诱导的RGC坏死性凋亡的分子机制。我们认为，IR诱导的视网膜节细胞TNF信号通路 促进形成持续产生活性氧（ROS）的正反馈回路， 其促进聚（ADP-核糖）聚合酶1（Parp 1）由于显著的氧化DNA而过度活化 损害RGC中显著的Parp 1过度激活介导ATP耗竭，导致随后的能量消耗 失败，这导致细胞功能障碍并最终导致RGC膜完整性的丧失（坏死）。我们 将采用广泛的生物化学、分子和细胞生物学技术以及动物模型， 在假设驱动机制中验证IR诱导RGC坏死性凋亡的分子机制 实验概述了以下具体目的：1）测试TNF信号传导促进IR的假设。 诱导的RGC坏死性凋亡; 2）检验IR诱导的RGC坏死性凋亡是由形成 以TNF信号依赖的方式持续产生ROS的正反馈回路; 3）测试 假设RGC坏死性凋亡是IR后Parp 1过度激活引起ATP耗竭的结果。因此， 由于对IR的治疗是有限的，部分原因是缺乏对导致IR的分子事件的理解， RGC死亡，更好地了解IR后RGC坏死性凋亡将导致新的治疗策略 重要和难以治疗的病症。

PROJECT SUMMARY Retinal ischemia-reperfusion (IR) injury is a common cause of visual impairment worldwide, affecting both patients' quality of life and functional status. Retinal ganglion cell (RGC) survival is critical for vision. However, these retinal neurons are exquisitely sensitive and many of them undergo necrosis and apoptosis after IR. Since RGC apoptosis is executed by programmed mechanisms and can be regulated, significant attention has been given to this type of cell death. At the same time, RGC necrosis did not receive nearly enough consideration, because it was viewed as an accidental and unregulated cellular event. We now know that necrosis, like apoptosis, can be executed by programmed mechanisms. This form of necrotic cell death is called necroptosis. Our published data and literature indicate that RGC necroptosis contributes to IR-induced retinal injury through direct loss of RGCs and induction of associated inflammatory responses. Therefore, since RGC necroptosis is executed by programmed mechanisms and can be regulated, this field of research is of great importance. However, the signaling cascades, which regulate IR-induced RGC necroptosis, still remain unknown. The long-term objective of this project is to identify the signaling cascades that regulate RGC necroptosis after IR. Based on our published data, preliminary studies and published literature, we proposed a molecular mechanism of IR-induced RGC necroptosis. We suggested that IR-induced Tnf signaling in RGCs facilitates formation of a positive-feedback loop for the sustained production of reactive oxygen species (ROS), which promotes poly (ADP-ribose) polymerase 1 (Parp1) over-activation due to significant oxidative DNA damage. Significant Parp1 over-activation in RGCs mediates ATP depletion, leading to subsequent energy failure, which results in cellular dysfunction and eventually in loss of RGC membrane integrity (necrosis). We will employ a wide range of biochemical, molecular and cell biological techniques as well as animal models to verify the proposed molecular mechanism of IR-induced RGC necroptosis in hypothesis-driven mechanistic experiments outlined in the following specific aims: 1) to test the hypothesis that Tnf signaling promotes IR- induced RGC necroptosis; 2) to test the hypothesis that IR-induced RGC necroptosis is promoted by formation of a positive-feedback loop for sustained ROS production in a Tnf signaling-dependent manner; 3) to test the hypothesis that RGC necroptosis is a result of ATP depletion caused by Parp1 over-activation after IR. Thus, since treatment for IR is limited in part because of a lack of understanding of the molecular events leading to RGC death, a greater understanding of RGC necroptosis after IR will lead to new therapeutic strategies for this important and difficult to treat condition.