Role and Regulation of a Distinct Optic Nerve Microglia Population in Glaucomatous Neuropathy - Glaucoma is chronic degeneration of the retina and optic nerves. However, the mechanisms by which ganglion cells in the retina communicate with cells in the myelinated optic nerve during ocular hypertension (OHT) are unclear. We recently discovered a resident neuroprotective retinal astrocyte- ganglion cell lipoxin circuit that is impaired during retinal stress, including OHT. Homeostatic astrocytes produce two endogenous lipoxins, Lipoxin A4 and Lipoxin B4, which act directly on retinal ganglion cells (RGCs). LXB4 is the most potent lipoxin in the retina, and it increases the survival and function of RGCs in OHT-induced neuropathy. To elucidate mechanism of actions of LXB4, we employed single-cell RNA sequencing, morphological characterization, and RNA sequencing to identify microglia as a novel cellular target for LXB4. We discovered that OHT induces distinct and temporally defined microglial functional phenotypes during the time course of sustained OHT in both the retina and myelinated optic nerve. Unexpectedly, microglial expression of CD74, a marker of disease-associated microglia (DAM), was only induced in the optic nerve but not in retinal microglia. LXB4 treatment during OHT shifted the optic nerve microglia toward homeostatic morphology and downregulated the expression of CD74. These findings identified microglia as a new LXB4 target in the optic nerve. LXB4 maintenance of optic nerve microglia and inhibition of the DAM phenotype are potential mechanisms for LXB4 neuroprotection. Hence, we hypothesized that the induction of CD74+ DAM in the optic nerve during OHT is a key signature of degeneration, and its regulation by LXB4 is neuroprotective mechanism against OHT-induced neuropathy. Aim-I during K99 phase will define how CD74+ DAM is regulated by LXB4 in the optic nerve during chronic OHT and identify targets and mechanisms specific to CD74+ DAMs in the optic nerve. For the R00 phase, in Aim-II, I intend to study and investigate how microglia in the remote distal myelinated optic nerve sense the insult to RGCs. In Aim III, I intend to study and investigate the role of optic nerve-specific DAMs driving the degeneration of axons. I will use spatial transcriptomics of optic nerve to define the cell -specific targets and differential proteomics approach to study changes in pathways and mediators in the optic nerves after OHT insult. To address the role of CD74+ DAM in the optic nerve during OHT, CD74 knockout mice will be used to study phenotypic, transcriptomic, and signal transduction regulation. In addition, to understand the crosstalk of glial cells in the optic nerve during OHT insult, I will use spatial transcriptomics based bioinformatic methods and electron microscopy to determine its effect on myelin health. Understanding the role of CD74+ DAM in the myelinated optic nerve during chronic OHT will deepen our knowledge of neurodegeneration mechanisms and open new paths for therapeutic interventions.
