KSEF RDE: Development of an Assay System for the Measurement of Beta-Secretase

The development of Alzheimer's Disease (AD) is marked by the progressive decline of cognitive function, including abnormalities of language, behavior, and memory [1]. As the amyloid precursor protein (APP) is trafficked through the secretory pathway, numerous cleavage events occur, releasing products into the extracellular space and vesicle lumens. One of these cleavage products is amyloidbeta (Aj3), which is derived from APP through sequential cleavage by two membrane bound enzymes, j3-secretase (SACE) and y-secretase. The progressive fibrillization and deposition of Aj3 within the brain is believed to be the primary causal factor in AD pathogenesis (2). Importantly, the activity and protein levels of SACE1 increase in brain both in normal aging and in AD [3-7). These findings therefore implicate {3-secretase activity as potentially critical for the development of APP-mediated disease. This proposal focuses on three distinct phases: (1) Development Commercially available SACE assay kits provide limited insight into beta-'secretase activity, as some endogenous enzymes cleave the SACE substrate. Many techniques used are, in fact, only semi-quantitative. This is unnecessary-it should be possible to develop a superior assay system that could be organized into a ready-made test kit. (2) Validation BACE1 and BACE2 show >65% homology. As currently available assays measure only total beta-secretase activity, it is necessary to develop an assay that can distinguish between these two secretase enzymes within various tissues. (3) Test and Compare We will investigate the veracity of this assay by examining tissue from mice and human cases and comparing our results with those from commercially available kits. Over the past year, our lab has gathered preliminary data indicating that the assay system we propose is feasible. Studies performed using this tool will lead to novel insights into the underlying processes that drive neurodegeneration, and will help in the future development of therapeutics.