Regulation of Diseases Associated Microglia in the Optic Nerve by Lipoxin B4 and Ocular Hypertension [scRNA-Seq]. Regulation of Diseases Associated Microglia in the Optic Nerve by Lipoxin B4 and Ocular Hypertension [scRNA-Seq]

The resident astrocytes-retinal ganglion cell lipoxin circuit is impaired during retinal stress that include exocytotoxic- and ocular hypertension-induced neuropathy. Two endogenous lipoxins (Lipoxin A4 and Lipoxin B4) produced by homeostatic astrocytes directly act on RGCs. LXB4 is the most potent lipoxin in the retina and directly increases RGC survival and function in ocular hypertension-induced neuropathy. Homeostatic roles and cellular targets of LXB4 in the retina and optic nerve are a critical gap in knowledge. Single-cell RNA sequencing was used to define cellular targets and signaling of LXB4 in the retina. For modeling neurodegeneration, sustained ocular hypertension was induced by silicone-oil injection in the anterior chamber of mouse eyes. For morphological characterization of microglia populations in the retina and optic nerve, we used MorphOMICs and pseudotime trajectory analysis. Bulk RNA sequencing of optic nerves was performed to characterize pathways and mechanism of action for LXB4. qPCR and immunohistochemistry were used for validation of transcriptomics data. Student’s t-test and one-way ANOVA were used to determine differences between experimental groups. Single Cell transcriptomic identified microglia as a primary target for LXB4 in the healthy retina. LXB4 downregulated genes that drive microglia environmental sensing and reactivity responses. Analysis of microglia function uncovered that ocular hypertension induces distinct, temporally defined and dynamic phenotypes in the retina and, unexpectedly, in the distal myelinated optic nerve. Microglial expression of CD74, a marker of disease-associated microglia (DAM) in the brain, was only induced in a unique population of optic nerve microglia but not the retina. Genetic deletion of lipoxin formation correlated with presence of a CD74 optic nerve microglia population in normotensive eyes optic, while LXB4 treatment during ocular hypertension shifted optic nerve microglia toward a homeostatic morphology and non-reactive state and downregulated expression of CD74. Furthermore, we identified a correlation between CD74 and phospho-PI3K (p-PI3K) expression levels in the optic nerve, that was reduced by LXB4 treatment. Results identify distal optic nerve microglial dynamic and reactive responses as a key feature of ocular hypertension induce neurodegeneration. Our findings establish microglia regulation as a new LXB4 cell target in the retina and optic nerve. LXB4 maintenance of optic nerve microglia homeostatic phenotype and inhibition of a disease-associated phenotype are potential mechanisms for LXB4 neuroprotection. Overall design: Mouse retinal cells were dissociated to single cells and 10x genomics based single cell transcriptomics was done after depleting the rod cells

驻留星形胶质细胞-视网膜神经节细胞(retinal ganglion cell, RGC)脂氧素信号通路在视网膜应激状态下受损，此类应激包括细胞毒性及眼高压诱导的神经病变。由稳态星形胶质细胞产生的两种内源性脂氧素——脂氧素A4（Lipoxin A4）与脂氧素B4（Lipoxin B4），可直接作用于RGC。脂氧素B4（LXB4）是视网膜中活性最强的脂氧素，可在眼高压诱导的神经病变中直接提升RGC的存活与功能。LXB4在视网膜与视神经中的稳态调控功能及细胞作用靶点，仍是当前研究的关键空白领域。本研究采用单细胞RNA测序（single-cell RNA sequencing）技术，明确了LXB4在视网膜中的细胞作用靶点与信号通路。为构建神经退行性疾病模型，本研究通过向小鼠眼前房注射硅酮油，诱导持续性眼高压。为表征视网膜与视神经中小胶质细胞群的形态学特征，本研究采用了MorphOMICs分析与拟时间轨迹分析（pseudotime trajectory analysis）。本研究对视神经开展批量RNA测序，以解析LXB4的作用通路与分子机制。本研究采用qPCR（定量聚合酶链反应）与免疫组织化学技术，对转录组学数据进行验证。本研究采用Student’s t检验与单因素方差分析（one-way ANOVA），以比较各组实验数据间的差异。单细胞转录组学分析显示，在健康视网膜中，小胶质细胞是LXB4的主要作用靶点。LXB4可下调介导小胶质细胞环境感知与活化反应的基因表达。对小胶质细胞功能的分析揭示，眼高压可在视网膜中诱导出具有时间特异性的独特动态表型；令人意外的是，在远端有髓鞘视神经中同样存在此类表型。作为脑部疾病相关小胶质细胞（disease-associated microglia, DAM）的标志物，CD74仅在视神经小胶质细胞的独特亚群中被诱导表达，而在视网膜小胶质细胞中则无此现象。脂氧素合成的基因敲除，可使正常眼压小鼠视神经中出现CD74阳性小胶质细胞亚群；而在眼高压模型中给予LXB4处理，则可将视神经小胶质细胞转向稳态形态与非活化状态，并下调CD74的表达。此外，本研究发现视神经中CD74与磷酸化PI3K（phospho-PI3K, p-PI3K）的表达水平呈正相关，而LXB4处理可削弱这一相关性。本研究结果证实，远端视神经小胶质细胞的动态活化反应是眼高压诱导神经退行性病变的关键特征。本研究结果确立了小胶质细胞调控作为LXB4在视网膜与视神经中的全新细胞作用靶点。LXB4维持视神经小胶质细胞稳态表型、并抑制疾病相关表型的作用，是其发挥神经保护功能的潜在分子机制。实验整体设计：将小鼠视网膜细胞解离为单细胞悬液，在去除杆状细胞后，采用基于10x Genomics平台的单细胞转录组学技术开展检测。