Mechanisms of toll-like receptor-mediated neurotoxicity in the ischemic retina

项目总结 视网膜组织的缺血性损伤是一种临床情况，经常导致视力障碍和失明， 影响患者的生活质量和功能状态。但更严格地理解 如果要有治疗视网膜疾病的新疗法，就需要缺血诱导的视网膜损伤。 发展起来的。先前，我们展示了Toll样受体4(TLR4)信号在视网膜中的有害作用 由缺血状态引发的炎症和损伤。由于TLR4信令由两个不同 信号级联，依赖于MyD88和Trif，我们单独评估了这些级联在 视网膜缺血症。现有证据似乎表明，TRIF和MyD88信号介导的效应 在缺血的视网膜组织中是视网膜特异的和不对称的。鉴于TRIF或MyD88的失活 导致缺血区视网膜炎症明显减轻，缺血区视网膜MyD88缺失 动物表现出比Trif缺乏动物的缺血视网膜更高的损伤水平。我们 还指出，缺血诱导的TRIF信号直接促进坏死性视网膜神经节细胞(RGC)的死亡。 此外，我们还证明了RGC坏死，这通过促进更多的 炎症，在某些情况下是可以调节的(称为“坏死性下垂”)。由于TRIF信号可以调节细胞 在目标1中，我们假设TRIF信号通过以下途径介导缺血诱导的视网膜损伤 促进RGC坏死性下垂。此外，依赖于Trif的信号不同于MyD88信号级联 它具有激活I型干扰素(IFN)信号的能力。先前的研究表明，TRIF通过激活干扰素， 激活caspase-11(Casp11)，进而直接或间接(通过NLRP3)介导细胞死亡 炎症体活性和白介素1b[IL1b]释放)。鉴于我们发现高水平的Casp11和 缺血视网膜节细胞的炎性小体活性，我们在目标2中假设缺血诱导的TRIF信号转导 介导Casp11的激活，进而直接或间接地介导RGC的死亡(通过升高 炎症反应)。最后，由于穆勒神经胶质细胞(MG)的活动与神经保护有关，几乎 在目标3中，我们假设视网膜中的每一种病理状态都是缺血诱导的MG特异性TLR4- 依赖的神经保护活性胜过神经毒性活性以促进缺血视网膜节细胞的存活。如果 提出的假设被证明是正确的，我们将能够解释TRIF和TRIF的不对称作用 缺血视网膜组织中的MyD88：而MyD88调节胶质细胞毒性(星形胶质细胞和小胶质细胞)和 缺血视网膜的神经保护作用，TRIF可直接激活上述两种现象 调节RGC死亡，从而促进比MyD88更显著的视网膜损伤。评估我们的 假设，我们将使用动物模型，并采用广泛的生化、分子和细胞生物学 技巧。通过完成这个项目，我们将为新的视网膜特异体组装一个智力基础 可以更有效地治疗缺血性视网膜疾病的治疗策略。

PROJECT SUMMARY Ischemic injury to retinal tissue is a clinical condition that frequently leads to visual impairments and blindness, affecting patients' quality of life and functional status. But a more rigorous understanding of the mechanisms of ischemia-induced retinal injury will be required if new therapies for the management of retinal disease are to be developed. Previously, we demonstrated the deleterious role of toll-like receptor 4 (Tlr4) signaling in retinal inflammation and damage triggered by ischemic conditions. Since Tlr4 signaling consists of two distinct signaling cascades, Myd88- and Trif-dependent, we individually evaluated the role of these cascades in ischemic retinae. Available evidence appears to indicate that the effects mediated by Trif and Myd88 signaling in ischemic retinal tissue are retina-specific and asymmetric. Whereas inactivation of either Trif or Myd88 resulted in significantly reduced inflammation in ischemic retinal tissue, ischemic retinae of Myd88-deficient animals demonstrated significantly higher levels of damage than ischemic retinae of Trif-deficient animals. We also noted that ischemia-induced Trif signaling directly facilitates necrotic retinal ganglion cell (RGC) death. Furthermore, we demonstrated that RGC necrosis, which exacerbates retinal injury by promoting more inflammation, can be regulated in some cases (termed “necroptosis”). Since Trif signaling can mediate cell necroptosis, we hypothesize in Aim 1 that Trif signaling mediates ischemia-induced retinal damage by promoting RGC necroptosis. In addition, Trif-dependent signaling differs from the Myd88 signaling cascade in its ability to activate type I interferon (IFN) signaling. It was previously shown that Trif, via its activation of IFN, activates caspase-11 (Casp11), which can in turn mediate cell death both directly and indirectly (via NLRP3 inflammasome activity and interleukin-1b [Il1b] release). In light of our finding of high levels of Casp11 and inflammasome activity in ischemic RGCs, we hypothesize in Aim 2 that ischemia-induced Trif signaling mediates Casp11 activation, which in turn mediates RGC death both directly and indirectly (via elevated inflammatory responses). Finally, since Müller glia (MG) activity is associated with neuroprotection in nearly every pathological condition in the retina, we hypothesize in Aim 3 that ischemia-induced, MG-specific, Tlr4- dependent neuroprotective activity prevails over neurotoxic activity to facilitate survival of ischemic RGCs. If the proposed hypotheses are found to be correct, we will be able to explain the asymmetric roles of Trif and Myd88 in ischemic retinal tissue: while Myd88 regulates glial toxicity (astrocytes and microglia) and neuroprotection (MG) in ischemic retinae, Trif activates both aforementioned phenomena while also directly mediating RGC death, thus promoting more significant retinal damage than Myd88. To evaluate our hypotheses, we will use animal models and employ a wide range of biochemical, molecular, and cell biological techniques. By completing this project, we will assemble an intellectual foundation for new retina-specific therapeutic strategies that can more effectively treat ischemic retinal diseases.