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，中风极大地增加了阿尔茨海默氏症的风险，因为它要么是一种诱发事件，要么是“触发”事件。细胞凋亡和Aa42的产生增加都与中风和头部创伤有关。虽然越来越多的知识表明脑血管疾病和阿尔茨海默病之间存在强烈的联系，但细胞凋亡和脑缺血在阿尔茨海默病中的作用仍不清楚。这项研究的中心假设是，与凋亡/caspase激活相关的条件(如脑缺血)增加了BACE蛋白水平和α-分泌酶活性，导致APP的淀粉样蛋白生成过程增强，从而导致AA毒性/产生的恶性循环。这一假说得到了我们体内和体外初步数据的有力支持，这些数据表明，在细胞凋亡过程中，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)以及大鼠脑缺血模型中总AA和Aa42水平的升高。我们还发现，凋亡过程中α-分泌酶活性的升高是半胱氨酸天冬氨酸酶激活后BACE蛋白稳定性增加的结果。用广谱caspase抑制剂zVAD抑制Caspase，可以阻止BACE的稳定和AA产量的增加。关于BACE的稳定机制，我们还发现BACE被溶酶体降解，参与BACE细胞内转运的适配蛋白GGA3是一种新的caspase底物，它在细胞凋亡时被切割。后者在体外细胞培养和脑缺血啮齿动物模型中均可观察到。这项拨款提案的目的是在体外和动物缺血模型中确定调节与凋亡/半胱氨酸酶激活相关的α-分泌酶活性和稳定性的分子机制。具体地说，我们建议：1：确定GGA3在正常条件下和在凋亡过程中对BACE稳定性的影响程度；2：确定在细胞凋亡过程中，哪种caspase(S)在BACE稳定(和增加AA生成)中起作用；3：确定caspase介导的BACE蛋白水平和α-分泌酶活性增加在多大程度上促进体内AA产生的增加。

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.