Molecular mechanisms of programmed necrosis in the ischemic retina

项目总结 视网膜缺血再灌注(IR)损伤是世界范围内视力损害的常见原因，影响到 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， 这些视网膜神经元非常敏感，其中许多在IR后会发生坏死和凋亡。 由于RGC的凋亡是由程序化的机制执行的，并且可以被调节，因此引起了极大的关注 都是这种类型的细胞死亡。同时，RGC的坏死也远远没有收到足够的资金 考虑，因为它被视为一个偶然的和不受监管的细胞事件。我们现在知道了， 像细胞凋亡一样，坏死也可以通过程序化的机制来实现。这种形式的坏死性细胞死亡 称为坏死性下垂。我们已发表的数据和文献表明，RGC坏死性下垂与IR有关 视网膜损伤，通过直接丢失视网膜节细胞和诱导相关的炎症反应。因此，既然 RGC坏死性下垂是由程序化的机制执行的，可以进行调节，这一领域的研究是 非常重要。然而，调节IR诱导的RGC坏死性下垂的信号级联仍然存在 未知。该项目的长期目标是确定调节RGC的信号级联 IR后坏死性下垂。基于我们已发表的数据、初步研究和已发表的文献，我们提出了一个 IR诱导RGC坏死性下垂的分子机制。我们认为IR诱导视网膜神经节细胞中的肿瘤坏死因子信号。 有助于形成用于持续产生活性氧物种(ROS)的正反馈回路， 它由于显著的DNA氧化作用而促进聚(ADP-核糖)聚合酶1(PARP1)的过度激活 损坏。视网膜节细胞中显著的PARP1过度激活介导了ATP的耗竭，导致随后的能量消耗 失败，导致细胞功能障碍，最终导致RGC膜完整性丧失(坏死)。我们 将采用广泛的生化、分子和细胞生物学技术以及动物模型来 从假说驱动机制验证IR诱导RGC坏死性下垂的分子机制 具体目标如下：1)验证肿瘤坏死因子信号促进胰岛素抵抗的假说。 2)检验IR诱导的RGC坏死下垂是通过形成促进的假说。 以肿瘤坏死因子信号依赖的方式持续产生ROS的正反馈循环；3)测试 假设RGC坏死性下垂是IR后PARP1过度激活引起的ATP耗竭的结果。因此， 由于对导致IR的分子事件缺乏了解，IR的治疗受到限制 RGC死亡，更好地了解IR后RGC坏死性下垂将导致新的治疗策略 既重要又难于治疗的疾病。

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.