ABSTRACT Amyloid-beta (A?)?the principal component of amyloid plaques in Alzheimer?s disease (AD)?is generated by sequential secretase cleavages of the amyloid precursor protein (APP), a type 1 transmembrane protein. Despite three decades of study, the precise subcellular locations of A? generation have remained elusive. This is important because not only is A? central to AD pathogenesis but reducing A? levels has been a major focus in the development of potential AD therapeutics. Determing the precise cellular locations of A? generation has been a major bottleneck because current model systems are incapable of tracking A? peptide in living cells. We have recently developed a novel APP construct which incorporates an unnatural amino acid in the extracellular A? segment of APP, enabling click-chemistry to attach a small molecule fluorophore to A? at the plasma membrane. Coincident attachment of distinct fluorescent proteins to intra- and extracellular regions of APP allows the real-time visualization of APP trafficking and A? generation in living cells. In this application we will exploit this model system to determine the impact of retromer sorting on APP trafficking and A? generation in engineered neural cell lines (Aim 1) and to determine the temporal-spatial dynamics of APP trafficking and A? generation within primary neurons (Aim 2). At the completion this grant, we will have a basic understanding of the itineraries of APP that lead to A? generation. We will also be able to examine these same pathways in primary neurons (or even iPSCs), and study how these A?-generating pathways may be altered by genetic variants or neuronal activity.