Mechanisms of proinflammatory glial activation in retinal degeneration - Project Summary / Abstract: The goal of this project is to define dysregulation in key immune checkpoints that regulate microglial activation, including TREM2-TYROBP-APOE signaling, which is associated with a common pro-inflammatory state, and how this contributes to progression of degeneration in age-related macular degeneration (AMD). To explore the hypothesis that proinflammatory microglia are a key component of the pathobiology of retinal degeneration in AMD, we will use CRISPR-Cas9 knockout of key immune checkpoints in human iPSC-derived microglia and expose them to multiple components of extracellular deposits that are characteristic of this disease (Aim 1). This will validate these pathways as potential targets for novel therapeutics aimed at limiting inflammation in AMD. In Aim 2, we will perform single-nucleus transcriptional profiling of retinal microglia from individuals with early and late stages of AMD, and those of controls. In Aim 3, we will investigate how transcriptional alterations in microglia are influenced by the pathologic tissue microenvironment by performing highly multiplexed spatial analysis of RNA and protein in retinal tissue affected by AMD-associated lesions using spatial co-indexing of transcriptomes and epitopes for multi-omics mapping by highly parallel sequencing (spatial-CITE-seq). Assaying transcriptional states in disease associated microglia in retina and within AMD lesions directly using human samples will provide a new paradigm to understand AMD and probe the largely unaddressed question of the role of microglia in AMD pathogenesis. It will also set the stage for further study of neuroinflammatory biology using AMD as a paradigm to discover principles with application to other neurodegenerative diseases. This mentored clinical scientist research career development award is designed to help Dr. Marcello DiStasio achieve his long-term career goal of an academic career with a focus on neuroinflammation in degenerative diseases of the retina. His Mentor, Dr. David A. Hafler, is a leader in the field of neuroimmunology and is committed to the scientific development and execution of the project. He is the William S. and Lois Stiles Edgerly Professor and Chairman, Department of Neurology and Professor of Immunobiology, Yale School of Medicine, and is the Neurologist-in-Chief of the Yale New Haven Hospital. Co-Mentor Dr. Brian P. Hafler is an Assistant Professor of Ophthalmology and Visual Science and of Pathology whose lab focuses on retinal disease using advanced genomics technologies. The environment at the Yale School of Medicine has a strong and established research program related to immunology and neuroscience, and includes the Center for Neuroinflammation, where the research will be conducted. The experience, knowledge, and skills gained through the research plan and career development activities will form the basis for future studies as Dr. DiStasio transitions to independence as a clinician-scientist.
