Mechanisms of microglial neuroinflammatory response in glaucoma

这项提案的目标是阐明小胶质细胞的作用，视网膜和视网膜的常驻免疫细胞。 青光眼的发病机制。青光眼是世界范围内不可逆失明的主要原因， 其标志是视网膜神经节细胞（RGC）的进行性丧失。在我们先前的工作中，我们发现， 青光眼中的小胶质细胞上调了与大脑共享的疾病相关分子标记 神经退行性变，并且在该信号传导途径中的分子的遗传或药理学靶向可以 防止青光眼小鼠模型中的RGC损失。此外，我们还发现， 凋亡神经元的吞噬可随后在没有高眼内压的情况下引起更多的RGC损失。 结果表明，小胶质细胞可以传播青光眼中的神经元损伤。使用RNAseq 从这些研究的数据集，我们现在已经确定了跨膜受体CD300lf作为一个关键的调解人， 青光眼中的小胶质细胞炎症反应CD300lf是一种磷脂酰丝氨酸受体， 抑制能力，可以结合到受损和凋亡细胞，并在大脑中，只表达由骨髓 细胞我们的初步数据表明：1）CD300 lf在两种小鼠视网膜小胶质细胞中上调， 青光眼模型; 2）CD300lf-/-动物在微珠青光眼模型中被保护免于RGC损失，尽管 升高的IOP;和3）与野生型小胶质细胞不同，CD300 lf-/-小胶质细胞不上调炎性细胞因子， 包括IL-1 β，在体内响应于受损/凋亡的神经元。根据这些数据，我们假设， CD300 lf是小胶质细胞炎症反应的关键调节因子，其加剧青光眼中的RGC损失。我们 将通过以下具体目标来解决这一假设： 1.确定小胶质细胞是否是介导CD300lf效应的关键髓样细胞亚群。 青光眼在这个目标中，我们将使用CD300lffl/fl条件性敲除来鉴定CD300lf是否通过以下方式损害RGC： 作用于小胶质细胞或其他骨髓细胞亚群（单核细胞、边界相关巨噬细胞）。 2：研究靶向CD300lf及其结合配偶体对炎性体信号传导的影响。我们 将评估CD300 lf或CD300 lf缺陷的骨髓细胞中的炎症反应和炎性小体功能， 其在细胞培养物和体内的结合伴侣。 3.探讨靶向CD300lf治疗青光眼的可能性。我们将评估 CD300lf中和抗体片段在青光眼小鼠模型中保护RGCs的作用， CD300 LF在青光眼患者死后眼睛中上调。 本提案中概述的研究将确定CD300lf信号在青光眼小胶质细胞中的作用， 最终目标是为这种常见的致盲疾病开发新的神经保护治疗方法。

The goal of this proposal is to elucidate the role of microglia, the resident immune cells of the retina and the brain, in the pathogenesis of glaucoma. Glaucoma is the leading cause of irreversible blindness worldwide, whose hallmark is progressive loss of retinal ganglion cells (RGCs). In our prior work we have found that microglia in glaucoma upregulate a disease-associated molecular signature that is shared with brain neurodegenerations, and that genetic or pharmacologic targeting of molecules in this signaling pathway can prevent RGC loss in a mouse model of glaucoma. Furthermore, we have found that microglia activated by engulfment of apoptotic neurons can subsequently cause more RGC loss in the absence of high intraocular pressure (IOP), demonstrating that microglia can propagate neuronal damage in glaucoma. Using the RNAseq datasets from these studies, we have now identified the transmembrane receptor CD300lf as a key mediator of microglial inflammatory response in glaucoma. CD300lf is a phosphatidylserine receptor with dual activating and inhibitory capacity that can bind to damaged and apoptotic cells and, in the brain, is expressed only by myeloid cells. Our preliminary data demonstrate that 1) CD300lf is upregulated in retinal microglia from two mouse glaucoma models; 2) CD300lf–/– animals are protected from RGC loss in the microbead glaucoma model despite elevated IOP; and 3) unlike wildtype microglia, CD300lf–/– microglia do not upregulate inflammatory cytokines, including Il-1, in response to damaged/apoptotic neurons in vivo. Based on these data, we hypothesize that CD300lf is a key regulator of the microglial inflammatory response that exacerbates RGC loss in glaucoma. We will address this hypothesis with the following Specific Aims: 1. Determine if microglia are the key myeloid cell subpopulation that mediates CD300lf effect in glaucoma. In this aim, we will use CD300lffl/fl conditional knockouts to identify whether CD300lf harms RGCs by acting in microglia or other myeloid cell subpopulations (monocytes, border-associated macrophages). 2: Investigate the effect of targeting CD300lf and its binding partners on inflammasome signaling. We will evaluate the inflammatory response and the inflammasome function in myeloid cells deficient in CD300lf or its binding partners in cell culture and in vivo. 3: Investigate the therapeutic potential of targeting CD300lf in glaucoma. We will assess the efficacy of CD300lf neutralizing antibody fragment in protecting RGCs in a mouse model of glaucoma and evaluate if CD300LF is upregulated in human postmortem eyes with glaucoma. The research outlined in this proposal will define the role of CD300lf signaling in microglia in glaucoma, with the ultimate goal of developing novel neuroprotective treatments for this common blinding disease.