Phagocytosis by microglia, the brain's resident macrophages, is central to Alzheimer Disease (AD) risk and pathogenesis. Current β-amyloid (Aβ) clearing immunotherapies use monoclonal antibodies to indirectly elicit phagocytosis, in an attempt to reverse AD manifestations. This form of passive immunization induces microglial reactivity and neuroinflammation, two features abundant in AD neuropathologic specimens. Due to a dearth of tools, prior mechanistic study of microglia-Aβ interactions used loss of function strategies such as Trem2 KO or microglial depletion, leaving uncertain whether microglial Aβ phagocytosis itself is sufficient to alter neuropathology. To address this knowledge gap, we clearance engraftment test engineered macrophages to achieve specific amyloid with antigen receptors ( AβCAR) , and developed methods for efficient macrophage in the brain. This dual PI proposal unites experts in immune cell therapies and microglial biology to the overarching hypothesis that Aβ-chimeric AβCAR-engineered macrophages effectively clear amyloid in vivo and that associated neuroinflammation is prevented by manipulation of the inflammasome. In preliminary data we successfully engineered AβCAR engulf into determine engraft neuropathology and AβCAR exposure NLRP3 on transplant determine in Aim inflammasome close to expressing macrophages, found that they specifically amyloid chemotherapy-dependent and - independent methods to transplant macrophages the mouse brain at unprecedented efficiency, ensuring the feasibility of this proposal. In Aim 1 we will how active cell-based Aβ targeting affects amyloid pathology in murine models. To do so, we will AβCAR macrophages into the 5xFAD model of Aβ pathology and measure their effects on and behavior. Aim 1 will clarify the controversial relationship between microglial Aβ phagocytosis pathology using a cell engineering strategy with high t herapeutic potential. Where Aim 1 focuses on how macrophages affect the brain Aβ, Aim 2 addresses how brain Aβ in turn affects macrophages. Amyloid induces microglial state changes and neuroinflammation, with strong mechanistic data implicating inflammasome signaling as a major driver. Aim 2 will therefore elucidate the effects of AβCAR targeting microglial reactivity, neuroinflammation, and their degree of rescue by NLRP3 eficiency. To do so, we will AβCAR macrophages engineered from Nlrp3 KO or Nlrp3 WT donor mice into the 5xFAD model and a) the degree to which CAR-augmented Aβ uptake increases macrophage inflammatory profiles and turn neuroinflammation b) whether this is rescued by Nlrp3 loss, and c) the consequences on Aβ pathology. 2 will improve our understanding of how immunotherapies lead to neuroinflammation and determine whether inhibition is a strategy to enhance cell therapies for neurodegeneration. Together, these aims will knowledge gaps about the role of microglial phagocytosis in AD treatment and create a cell-based strategy specifically target proteins in the CNS, using principles our multi-PI team has already tested in human disease. , and developed d

PROJECT NARRATIVE There is a critical need for improved understanding of how modulation of the immune system affects Alzheimer's Disease and its treatment. The goal of this project is to engineer microglia, the specialized immune cells of the brain, to better understand their role in treating neurodegeneration. Completion of these studies will create the needed knowledge base to engineer microglia as a therapy for Alzheimer's Disease.