Neovascular age-related macular degeneration (AMD) is a neuroinflammatory disease that is a leading cause of blindness in the elderly. While the involvement of photoreceptors in AMD has been well established, our research will study the less understood role that inflammation plays in the development of AMD. Our proposal will address this knowledge gap and yield a detailed understanding of inflammation-associated AMD pathogenesis in humans, in addition to the identification of potential therapeutic targets and treatments for AMD. Our access to human tissue with advanced neovascular AMD from the Yale Rapid Autopsy Service and single-cell RNA sequencing expertise will allow us to perform studies that profile the transcriptome in activated innate immune cells. Our preliminary data indicate that the critical inflammatory pathways reside in microglia and monocyte-derived macrophages. Our overarching hypothesis is that functional changes in the innate system influence neovascularization in AMD, and these changes may be targeted to halt disease progression. To explore this hypothesis, we propose the following two specific aims. In Aim 1, we will perform highly parallel single-nucleus transcriptional profiling with a novel enrichment technique for glia from human eyes with exudative AMD. The primary goal is to define and interrogate the molecular signature of microglia and macrophages. Our preliminary data revealed activated microglia in AMD with secretion of the proinflammatory cytokine interleukin-1b. We hypothesize that there is as upregulation of inflammatory molecules, which is associated with choroidal neovascularization in AMD. In Aim 2, we will target the pro- inflammatory cytokines that regulate activation of reactive Müller glia in AMD to identify targetable pathways to reverse the chronic inflammation in disease. Based on preliminary data, our targets will include the IL-1b, IL-10, and IL-17 pathways as well as additional ones identified in Aim 1. We hypothesize that inflammatory molecules are critical for the transformation of homeostatic Müller glia to a reactive, pro-angiogenic state in AMD. The proposed research plan will provide unprecedented insight into the molecular mechanisms of AMD progression and has significant potential to identify novel therapeutic targets for drug discovery. We anticipate that our work will lead to the development of the first effective therapeutic approaches targeting inflammation, thereby improving the quality of life for individuals suffering from neovascular AMD.