Biological Significance of Oxidized Neprilysin

描述（由申请人提供）：本研究的长期目标是了解脑啡肽酶氧化修饰对衰老和阿尔茨海默病（AD）中淀粉样蛋白沉积的生物学意义。脑实质和血管中的淀粉样蛋白沉积是老年人的常见现象，也是AD的诊断标志之一。在衰老和AD大脑中过量β-淀粉样蛋白（AB）沉积的两种可能的解释是过度生产和降解减少。虽然参与AB生产的机制已被广泛研究，并已证实AB的过度生产在家族性AD病例的发病机制中起着关键作用，但到目前为止，很少有证据表明，大脑AB生产增加在衰老和散发性AD中是重要的。最近，AB降解的作用已经越来越多地被研究，并且已经描述了具有一系列降解AB的能力的几种酶。其中，脑啡肽酶（NEP）被认为是AB降解的关键酶。以往的研究表明，NEP降低可能有助于AD中AB的积累。我们小组最近的数据表明，大脑NEP在AD和衰老中都受到氧化修饰。为了理解氧化的NEP的生物学意义，了解氧化是否降低NEP酶活性是非常重要的。了解NEP的HNE修饰位点以设计可以阻断NEP的氧化修饰的治疗试剂也是重要的。我们的工作假设是，最初的淀粉样蛋白积累将导致HNE的生产与渐进的抑制性NEP，开始一个前馈循环增加淀粉样蛋白积累，氧化应激，和NEP抑制。为了验证这一假设，作为初始步骤，我们在RO 3提案中提出了三个具体目标：1。目的：1.确定外源性HNE或人工合成AB诱导的内源性HNE能否修饰培养细胞中表达的人NEP。发现在培养细胞中表达的HNE修饰的NEP是否具有降低的酶活性。3.确定改变NEP活性的关键HNE修饰位点。我们的目标是通过这种RO 3机制为未来的RO 1应用开发足够的初步数据，以进一步探索动物模型和人类的这一假设。

DESCRIPTION (provided by applicant): 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.