Accumulation of b- amyloid is one of the hallmarks of Alzheimer's disease (AD). This accumulation could be the result of excess production of amyloid or impaired clearance. Genetic studies are consistent with a role for b- amyloid plaque in the onset of some familial AD, but the mechanisms of causation and the role of plaque in the onset of sporadic AD remain uncertain. Microglia, the major inflammatory cells of the CNS, are one of the cell types that is associated with plaque, and there is suggestive evidence that a process similar to chronic inflammation may occur in AD. The goal of this project is to characterize the interactions between b-amyloid and microglia. As the major scavenger cells in the brain, microglia would be expected to play a role in clearance of amyloid, as well as in generating an inflammatory response. As a first step in the characterization, the receptors involved in interactions between b-amyloid and microglia will be identified. This will be done using biochemical and fluorescence methods to measure binding and uptake. Specific inhibitors of known receptors will be tested for their ability to block uptake. The mode of uptake and degradation (e.g., endocytosis or phagocytosis) will be studied by optical microscopy and electron microscopy. The ability of amyloid plaque to initiate signaling responses in microglia will also be studied. Early responses will include activation of phagocytosis and [Ca2+] signaling. These will be measured in single cells by optical microscopy. Later responses studied will be release of IL-1 and complement components, which would be indicative of an inflammatory reaction. Initial studies will be carried out with plaque formed from synthetic peptides. Other protein components of AD amyloid plaques will be incorporated into the plaques to see if they affect receptor binding or activation of signaling. These include various isoforms of apo- lipoprotein E (Apo-E2, E3, and E4) as well as complement. After preliminary studies using these reconstituted plaques and mouse microglia, studies will be carried out with human microglia and plaque from human AD brains. These studies should provide fundamental information about the receptors that bind amyloid and their effects in activating microglia. All of these studies will follow methods used previously by the P.I. for the study of endocytosis, phagocytosis, [Ca2+]i signaling, and motility in macrophages.
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