Alzheimer's disease (AD) is a devastating and progressive disease without a cure. Long thought to be a consequence of AD, neuroinflammation is now recognized to be a key early event in the development of AD. Neuroinflammation in AD is predominately driven by innate immunity mediated by microglia. Aberrant neuroinflammatory response activates immune cells and releases enzymes and factor to generate oxidative stress that can directly damage neurons and axons, cause demyelination, and break down the blood-brain barrier (BBB), and has been found in patients with early stage AD. Current knowledge on these cells and their functions has been derived from indirect observations utilizing in vitro assays that prevent longitudinal tracking in living animals and patients. While imaging could potentially track inflammation in vivo, no imaging agent currently available can specifically report on microglia and characterize its functional phenotype in vivo. Thus, there is an unmet need to develop imaging agents that can specifically target different types of microglia, especially damaging-type microglia that participate in the AD pathogenesis. The novelty of this proposal is in 1) we have identified multiple potential markers that are unique to damaging microglia from the parent award, 2) at least four of these markers have been found to be associated with AD, and 3) we have developed prototype imaging agents for two of these targets that will now be tested and validated for AD. We hypothesize that the targets identified in stroke from the parent award can also be used to identify damaging microglia in AD. The goal of this administrative supplement proposal is to validate the stroke-derived imaging targets and potentially identify new imaging targets for damaging microglia in AD. The aims are 1A) identify and validate potential imaging targets for damaging microglia in AD, and 1B) validate potential imaging agents for damaging microglia in AD. Using high-throughput mass spectrometry proteomics, we will profile proteins from microglia subtypes and compare to macrophage subpopulations to verify existing targets found from the parent project and identify new unique targets for damaging microglia in AD. We will use two different mouse models of AD: 1) 5xFAD (β-amyloid pathology) and 2) rTg4510 (tau/neurofibrillary tangles pathology). We will also test using human cells obtained from a human 3D AD cell culture model that captures key features of human AD (β- amyloid, neurofibrillary tangles, neuroinflammation, and neurodegeneration). This supplement crossing stroke and AD will also provide a unique opportunity to observe how microglia are altered between these different diseases. We expect this supplement will enable future grants to develop and validate in vivo specific imaging agents to target damaging microglia in AD, and enable future studies to better understand the roles these microglia play in the development of AD and potentially other neurodegenerative disea...