PROJECT SUMMARY/ABSTRACT Alzheimer’s Disease (AD) is the most common form of dementia and is a neurodegenerative disease characterized by the accumulation of toxic amyloid beta (Aβ) and subsequent aggregation of hyperphosphorylated tau. Microglia are the resident immune cells of the central nervous system, which function to support neurons, remove debris, and modulate neuronal viability, among other functions. Signaling through the CX3CR1/CX3CL1 axis between microglia and neurons modulates microglial function and reactivity, and previous studies have shown that communication through this axis is essential in the maintenance of homeostatic conditions within the central nervous system, while disruptions have been associated with neurodegeneration. The microglial CX3CR1 receptor interacts selectively with CX3CL1 secreted by neurons to modulate inflammation, with previous studies demonstrating that gene variants in CX3CR1 are associated with neurodegeneration observed in AD. However, the mechanisms by which affected microglia lead to neurodegeneration in AD remain unclear, and these prior studies have primarily utilized murine models. While many advancements in the understanding of AD pathogenesis have been established through modeling the disease in mice, these models have failed to produce a clinically relevant therapeutic for AD patients. As such, there is a critical need to develop complimentary models that may account for at least some of these species-specific differences. Human induced pluripotent stem cells (iPSCs) can be differentiated into all cell types of the brain, with the possibility to establish novel co-culture models to accurately assess interactions between critical cell types, including the analysis of disease-associated gene variants and how they lead to neurodegeneration. Thus, the central goal of this application is to utilize iPSCs to investigate the role of the CX3CR1 V249I variant in microglial function and whether this variant induces a neurodegenerative phenotype in neurons. To accomplish this, in Aim 1, I will investigate the impact of the CX3CR1 V249I variant on iPSC-derived microglia alone by assessing features including inflammatory signaling, phagocytosis, and RNA sequencing. In Aim 2, I will determine whether interactions with V249I microglia mediate altered phenotypes and function in neurons using both 2D and 3D co-culture systems, as well as how neurons may further modulate disease phenotypes in microglia. I hypothesize that the CX3CR1 V249I variant will cause a dysfunctional phenotype in microglia, and that this will mediate phenotypic and functional changes to neurons in either a cell autonomous or non-cell autonomous manner. It is expected that this work has the potential to identify mechanisms by which a genetic variant modulates neurodegenerative pathology related to AD. Taken together, this proposal provides an innovative approach to investigating the pathophysiology of genetic variants related to AD in a nov...