Project description: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.

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Dysregulation of Neuroprotective Lipoxin Pathway in Astrocytes in Response to Cytokines and Ocular Hypertension

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