Mechanisms of microglial neuroinflammatory response in glaucoma

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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.