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

项目概要 视网膜组织缺血性损伤是一种经常导致视力障碍和失明的临床病症， 影响患者的生活质量和功能状态。但更严格地了解其机制 如果要开发治疗视网膜疾病的新疗法，就需要缺血引起的视网膜损伤。 开发的。之前，我们证明了 Toll 样受体 4 (Tlr4) 信号在视网膜中的有害作用 缺血性疾病引发的炎症和损伤。由于 Tlr4 信号由两个不同的 Myd88 和 Trif 依赖性信号级联，我们单独评估了这些级联在 视网膜缺血。现有证据似乎表明 Trif 和 Myd88 信号传导介导的作用 缺血性视网膜组织中的视网膜特异性和不对称性。而 Trif 或 Myd88 失活 导致缺血性视网膜组织、Myd88 缺陷的缺血性视网膜中的炎症显着减少 与 Trif 缺陷动物的缺血性视网膜相比，这些动物表现出明显更高水平的损伤。我们 还指出，缺血诱导的 Trif 信号传导直接促进坏死性视网膜神经节细胞 (RGC) 死亡。 此外，我们证明 RGC 坏死会通过促进更多的视网膜损伤而加剧视网膜损伤。 炎症，在某些情况下可以得到调节（称为“坏死性凋亡”）。由于 Trif 信号传导可以介导细胞 坏死性凋亡，我们在目标 1 中假设 Trif 信号通过以下方式介导缺血引起的视网膜损伤： 促进 RGC 坏死性凋亡。此外，Trif 依赖性信号传导与 Myd88 信号级联的不同之处在于 它能够激活 I 型干扰素 (IFN) 信号传导。之前的研究表明，Trif 通过激活 IFN， 激活 caspase-11 (Casp11)，进而直接或间接（通过 NLRP3）介导细胞死亡 炎症小体活性和白细胞介素 1b [Il1b] 释放）。鉴于我们发现高水平的 Casp11 和 缺血性 RGC 中的炎症小体活性，我们在目标 2 中假设缺血诱导的 Trif 信号传导 介导 Casp11 激活，进而直接和间接介导 RGC 死亡（通过升高 炎症反应）。最后，由于穆勒胶质细胞 (MG) 活性几乎与神经保护有关 对于视网膜的每一种病理状况，我们在目标 3 中假设缺血引起的、MG 特异性的、Tlr4- 依赖性神经保护活性优于神经毒性活性，有利于缺血性 RGC 的存活。如果 如果发现所提出的假设是正确的，我们将能够解释 Trif 和 缺血性视网膜组织中的 Myd88：Myd88 调节神经胶质细胞毒性（星形胶质细胞和小胶质细胞）和 Trif 可以激活缺血性视网膜中的神经保护（MG），同时还可以直接激活上述两种现象 介导 RGC 死亡，从而导致比 Myd88 更严重的视网膜损伤。来评估我们的 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 技术。通过完成这个项目，我们将为新的视网膜特异性奠定知识基础 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.