Project Summary/Abstract Recently, multiple human genetic studies have identified the critical role of the immune system in the pathogenesis of Alzheimer’s disease (AD). For example, microglia phagocytose amyloid beta (Abeta) and regulate brain immune function by secreting cytokines and chemokines. Because previous studies have suggested both protective and detrimental effects of microglial activity in AD, how microglial AD risk genes, such as PU.1, affect microglial function still remains unclear. Therefore, investigating the role of AD genetic risk factors in microglia will provide critical insight into the pathobiology of the disease, potentially revealing key regulators of underlying disease mechanisms and novel therapeutic targets. An AD genetic risk factor, PU.1, is a critical transcription factor selectively expressed in microglia in the brain. We hypothesize that PU.1 affects several transcription pathways, Abeta metabolism and other AD-related pathologies by regulating their immune function. To test this hypothesis, we will apply several innovative technologies, including single-cell RNA-sequencing, quantitative proteomics, super-resolution microscopy, human-induced pluripotent stem cell-derived microglia and neurons, multi-electrode arrays, electrophysiology, and the simultaneous Positron Emission Tomography- Magnetic Resonance Imaging, in collaboration of multiple collaborators with extensive experience in these methods. In aim 1, we will determine how downregulating PU.1 affects microglial and neuronal phenotypes and perform unbiased transcriptomic and proteomic analyses to identify key downstream regulators. In aim 2, we will investigate the functional interaction between microglia and neurons using human induced pluripotent stem cells after regulating PU.1 expression. In aim 3, we will terminate how microglial PU.1 affects AD phenotypes when it is regulated before and after the onset of amyloid pathology.