Role of BACE stabilization in Alzheimer's Disease (AD)

描述（申请人提供）：衰老、遗传因素和头部创伤是阿尔茨海默病（AD）的主要危险因素。此外，中风显著增加了阿尔茨海默病的风险，作为一个沉淀或“触发”事件。细胞凋亡和Aa42产生增加都与中风和头部创伤有关。虽然有越来越多的知识表明脑血管疾病和阿尔茨海默病之间有很强的关联，但细胞凋亡和脑缺血在阿尔茨海默病中的作用仍不清楚。该研究建议的中心假设是与细胞凋亡/半胱天冬酶激活相关的条件（例如脑缺血）增加BACE蛋白水平和α-分泌酶活性，导致APP的淀粉样蛋白形成加工的增强，从而导致Aa毒性/产生的恶性循环。我们的体内和体外初步数据有力地支持了这一假设，这些数据表明，在细胞凋亡期间α-分泌酶水平和活性增加，导致各种细胞类型（包括原代神经元）和大鼠和小鼠脑缺血模型中总Aa和Aa42水平升高。我们还发现，细胞凋亡过程中α-分泌酶活性的升高是胱天蛋白酶激活后BACE蛋白稳定性增加的结果。通过用广谱半胱天冬酶抑制剂zVAD处理的半胱天冬酶抑制能够防止BACE的稳定和Aa产生的增加。关于稳定化机制，我们还发现BACE被溶酶体降解，并且GGA 3（一种参与BACE细胞内运输的衔接蛋白）是一种新的半胱天冬酶底物，其在细胞凋亡期间被裂解。后者在体外细胞培养物和啮齿动物脑缺血模型中均观察到。本研究的目的是确定调节与细胞凋亡/半胱天冬酶激活相关的α-分泌酶的活性和稳定性的分子机制，包括体外和缺血动物模型。具体而言，我们建议：一曰：确定GGA 3在正常条件下和细胞凋亡期间影响BACE稳定性的程度; 2：确定哪种半胱天冬酶在细胞凋亡期间BACE稳定（和Aa生成增加）中发挥作用; 3：确定半胱天冬酶介导的BACE蛋白水平和α-分泌酶活性增加在体内有助于Aa生成增加的程度。

Description (provided by the applicant): Aging, genetic factors and head trauma are major risk factors for Alzheimer's disease (AD). Additionally, stroke significantly increases the risk of Alzheimer's disease, operating as either a precipitating or "triggering" event. Apoptosis and increased Aa42 production have both been associated with stroke and head trauma. While there is an increasing body of knowledge indicating a strong association between cerebrovascular disease and Alzheimer's disease, the role of apoptosis and cerebral ischemia in Alzheimer's disease remains unclear. The central hypothesis of this research proposal is that conditions associated with apoptosis/caspase activation (e.g cerebral ischemia) increase BACE protein levels and a-secretase activity resulting in a potentiation of the amyloidogenic processing of APP leading to a vicious cycle of Aa toxicity/production. This hypothesis is strongly supported by our in vivo and in vitro preliminary data showing that a-secretase levels and activity are increased during apoptosis, leading to elevated total Aa and Aa42 levels in a variety of cell types (including primary neurons), and cerebral ischemia models in rats and mice. We have also discovered that the elevated activity of a-secretase during apoptosis is the result of increased protein stability of BACE following caspase activation. Caspase inhibition by treatment with zVAD, a broad spectrum caspase inhibitor is able to prevent the stabilization of BACE and the increase in Aa production. Regarding mechanism of stabilization, we have also discovered that BACE is degraded by the lysosomes and that GGA3, an adaptor protein involved in BACE intracellular trafficking, is a novel caspase substrate that it is cleaved during apoptosis. The latter was observed both in in vitro cell cultures and in vivo in rodent models of cerebral ischemia. The objective of this grant proposal is to determine the molecular mechanisms that regulate the activity and stability of a-secretase associated with apoptosis/caspase activation both in vitro and in animal models of ischemia. Specifically, we propose: 1: To determine the extent to which GGA3 affects BACE stability under normal conditions and during apoptosis; 2: To determine which caspase(s) play a role in BACE stabilization (and increased Aa generation) during apoptosis; 3: To determine the extent to which caspase-mediated increase in BACE protein levels and a-secretases activity contributes to increased Aa production in vivo.