The deposition of the small ABeta peptide as insoluble Beta-amyloid plaque in the brain parenchyma is an invariant feature of Alzheimer's disease (AD) and is thought to be central to the pathogenesis of the disease. Strategies to reduce ABeta generation by neurons or increase ABeta clearance from the brain hold the greatest potential as therapeutic interventions for the treatment or prevention of AD. ABeta is generated in two steps by the Beta- and gamma-proteolytic cleavages of the amyloid precursor protein (APP). BACE, a transmembrane aspartyl protease, accounts for most of the Beta-cleavage activity within cells, and inhibitors of this activity are particularly appealing AD therapeutics. We propose a single Specific Aim in which we will develop and characterize sensitive and highly specific sandwich ELISAs for cell-associated APP metabolites (APP holoprotein, Beta-cleaved carboxyl-terminal APP fragments, other APP carboxy-terminal fragments). These ELISAs will permit the rapid screening on living cells of compounds in order to identify those compounds that specifically reduce APP Beta-cleavage. Additionally, these ELISAs will be streamlined and scaled to permit their use in the high-throughput screening of B-cleavage inhibitors using living cells. Unfortunately, high-throughput drug screening on living cells typically generates an overwhelming number of false-positive hits because many compounds are simply toxic, and nonspecific toxicity is likely to reduce the target activity. For this reason, high-throughput screening of living cells is rarely performed when the desired outcome is a reduction in a particular cellular activity. This is particularly a hurdle for AD drug discovery, because compounds are sought that reduce ABeta generation by a neuron. This cell-based screening method, however, will allow for the elimination of most false-positive hits due to nonspecific toxicity while detecting particularly informative cellular metabolites along the pathway to AB generation. Overcoming the false-positives associated with toxicity makes high-throughput cell-based screening a practical, cost-effective, and appealing approach. Additionally, once a compound is identified as effective in a cell-based system it has a much greater probability of in vivo efficacy than a compound identified, for example, using a purified enzyme screen.