The long-term objective of this study is to understand the biological significance of oxidative modification of neprilysin on amyloid deposition in aging and Alzheimer's disease (AD). Amyloid deposition in the brain parenchyma and in vessels is a common phenomenon with advanced age and one of the diagnostic hallmarks for AD. Two possible explanations for excessive beta-amyloid (AB) deposition in aging and AD brains are overproduction and decreased degradation. Although the mechanisms involved in AB production have been extensively studied and the excessive production of AB has been confirmed to play a critical role in the pathogenesis of familial AD cases, there is little evidence so far to suggest that increased brain AB production is important in aging and sporadic AD. Recently, the role of AB degradation has been increasingly studied and several enzymes have been described with a range of abilities to degrade AB. Among them, neprilysin (NEP) has been considered by some a pivotal enzyme for AB degradation. Previous studies have shown that decreased NEP may contribute to the accumulation of AB in AD. Recent data from our group indicated that brain NEP is subject to oxidative modification in both AD and aging. To understand the biological significance of oxidized-NEP it is very important to know if oxidization decreases NEP enzymatic activity. It is also important to know the HNE modification site(s) of NEP to design therapeutic reagents that can block oxidative modification of NEP. Our working hypothesis is that initial amyloid accumulation will lead to HNE production with progressive inhibitory NEP, starting a feed-forward cycle of increasing amyloid accumulation, oxidative stress, and NEP inhibition. To test this hypothesis, as initial steps we propose three specific aims in this RO3 proposal: 1. To determine if human NEP expressed in cultured cells can be modified by exogenous HNE or endogenous HNE induced by synthetic AB; 2. To discover if HNE-modified NEP expressed in cultured cells has decreased enzymatic activity. 3. To identify critical HNE-modification sites which alter NEP activity. Our goal through this RO3 mechanism is to develop enough preliminary data for a future RO1 application that will further explore this hypothesis in animal models and in humans. ? ? ?
