ASTROCYTIC BACE1 EXPRESSION AND BETA-AMYLOID PEPTIDE GENERATION

淀粉样斑块是阿尔茨海默病（AD）的主要病理标志。β-淀粉样肽（Abeta）是淀粉样斑块的主要成分，有证据表明Abeta是AD发病机制的核心。Abeta由淀粉样前体蛋白（APP）通过两种蛋白酶（β-和γ-分泌酶）的连续切割产生。β-分泌酶是一种新的天冬氨酸蛋白酶，命名为BACE 1，是本课题组首次克隆和鉴定的。BACE 1启动Abeta的形成，因此是治疗AD的关键药物靶标。最近一代的BACE 1-/-小鼠基因打靶在我们的小组已经验证了BACE 1作为真正的β-分泌酶在体内。 在AD中，淀粉样蛋白斑块与涉及活化的神经胶质的局灶性炎症反应相关。 炎症标志物在AD患者和淀粉样斑块形成APP转基因小鼠的大脑中显著升高。IL-1 β是AD过程中释放的主要细胞因子，最近的研究表明，IL-1 β增加了培养的星形胶质细胞中APP的合成。此外，在APP转基因小鼠中，斑块周围活化的星形胶质细胞表达BACE 1。这些数据表明，活化的星形胶质细胞表达APP底物和BACE 1酶，因此可能产生大量的Abeta。我们的假设是淀粉样斑块周围的活化星形胶质细胞通过在AD炎症过程中产生Abeta而加剧斑块的发展。非甾体抗炎药（NSAID）可降低AD风险，NSAID布洛芬可抑制APP转基因小鼠中淀粉样斑块的形成，这一假说得到了证据的支持。该项目的长期目标是了解活化的星形胶质细胞在Abeta产生和淀粉样斑块发展中的作用。具体目标如下：（1）确定活化的星形胶质细胞中BACE 1和APP水平是否增加，如果是，活化的星形胶质细胞是否表现出增加的Abeta产生并有助于淀粉样蛋白沉积; 2.）的情况。 确定IL-1 β是否导致体外和体内星形胶质细胞和神经元中APP和BACE 1合成和Abeta产生增加，并促进体内淀粉样蛋白沉积。 我们将使用培养的小鼠星形胶质细胞和转基因和基因敲除小鼠作为本项目的主要模型系统。我们的分析方法将包括分子（北方，西方，免疫沉淀，ELISA）和细胞（免疫组织化学/光学显微镜，免疫荧光/共聚焦显微镜）技术的组合。重要的是，我们将开发新的APP转基因模型，并在该项目中使用Griffin及其同事的IL-1 β颗粒植入模型。我们预计，这些研究将提供关键的洞察星形胶质细胞和BACE 1在Abeta生成和淀粉样蛋白沉积的作用，是中央AD病理生理机制。

Amyloid plaques are a major pathological hallmark of Alzheimer's disease (AD). The beta-amyloid peptide (Abeta) is the main component of amyloid plaques, and evidence suggests that Abeta is central to AD pathogenesis. Abeta is generated from the amyloid precursor protein (APP) via sequential cleavage by two proteases, the beta- and gamma-secretases. The beta- secretase, a novel aspartic protease termed BACE1, was initially cloned and characterized in our group. BACE1 initiates the formation of Abeta and therefore is a key drug target for the treatment of AD. The recent generation of BACE1-/- mice by gene targeting in our group has validated BACE1 as the authentic beta-secretase in vivo. In AD, amyloid plaques are associated with a focal inflammatory response that involves activated glia. Inflammatory markers are dramatically elevated in the brains of AD patients and amyloid plaque-developing APP transgenic mice. IL-1beta is a major cytokine released during AD, and recent studies demonstrate that IL-1beta increases the synthesis of APP in cultured astrocytes. Moreover, in APP transgenic mice, activated astrocytes around plaques express BACE1. These data suggest that activated astrocytes express both APP substrate and BACE1 enzyme and thus may produce significant amounts of Abeta. Our hypothesis is that activated astrocytes surrounding amyloid plaques exacerbate plaque development by generating Abeta during AD inflammation. This hypothesis is supported by evidence that nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the risk of AD, and that the NSAID ibuprofen inhibits amyloid plaque development in APP transgenic mice. The long-term goal of this project is to understand the role of activated astrocytes in Abeta production and amyloid plaque development. The Specific Aims are the following: 1.) Determine whether BACE1 and APP levels are increased in activated astrocytes and, if so, whether activated astrocytes exhibit increased Abeta production and contribute to amyloid deposition; 2.) Determine whether IL-1beta causes increases in APP and BACE1 synthesis and Abeta production in astrocytes and neurons in vitro and in vivo, and facilitates amyloid deposition in vivo. We will use cultured mouse astrocytes and transgenic and knockout mice as our main model systems for this project. Our analytical approaches will include a combination of molecular (Northern, Western, immunoprecipitation, ELISA) and cellular (immunohistochemistry/light microscopy, immunofluoescence/confocal microscopy) techniques. Importantly, we will develop novel APP transgenic models and use the IL-1beta pellet implantation model of Griffin and colleagues in this project. We anticipate that these studies will provide critical insight into the role of astrocytes and BACE1 in Abeta generation and amyloid deposition, mechanisms that are central to AD pathophysiology.