Mechanisms of BACE1 Up-regulation in Response to Energy Deprivation

描述(由申请人提供)：β位点APR裂解酶(BACE1)是产生淀粉样蛋白(3，AP)的限速酶，淀粉样蛋白是阿尔茨海默病(AD)患者大脑中老年斑的主要成分。有证据表明BACE1在转录后水平因能量抑制而上调，研究表明AD大脑中的糖代谢降低，以及BACE1蛋白在死后AD大脑中水平升高的观察结果都支持这样的假设，即在大脑中缺乏能量的情况下，BACE1在转录后水平的上调可能在一定程度上导致了散发性阿尔茨海默病(SAD)中AP的积聚。本研究的目的是在体外和体内研究能量剥夺导致BACE1表达上调的特定转录后机制和信号通路(S)。对293细胞的初步研究表明，BACE1蛋白在能量剥夺后的增加是由BACE1 5‘-非翻译区(UTR)介导的。首先，我们将确定在培养的神经元中，是否有类似的机制控制BACE1蛋白水平，以应对能量剥夺。然后，荧光素酶报告分析将被用来确定在缺糖条件下293细胞和培养的神经元中BACE1蛋白的增加是否与BACE1的5‘端非编码区有关。在缺乏BACE1 5‘UTR的转基因小鼠中，急性药物诱导代谢应激将被用来在体内证实这一机制。初步实验表明，在能量剥夺条件下，真核细胞翻译起始因子2的α亚基(ElF2pha)的磷酸化可能是BACE1蛋白转录后增加的中介。我们将使用293个细胞和体外剥夺葡萄糖的神经元，以及遭受急性和慢性能量剥夺的小鼠的大脑来测试已知的磷酸化elF2pha的激酶的激活。然后，我们将确定通过thapsigargin处理的药物诱导elF2pha磷酸化是否导致BACE1蛋白在体外转录后增加。最后，为了建立该途径作为潜在的AD药物靶点，我们将在体外抑制elF2pha的磷酸化，以确定在葡萄糖缺乏的条件下是否可以阻断BACE1蛋白的上调。预计阿尔茨海默病的发病率将增加，目前缺乏有效的治疗方案，这使得开发更有效的治疗阿尔茨海默病的新药成为公共卫生的优先事项。这些目标的完成将为开发预防性AD疗法确定新的药物靶点。

DESCRIPTION (provided by applicant): Beta-site APR cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid (3 (Ap), the primary constituent of senile plaques in the brains of Alzheimer's disease (AD) patients. Evidence that BACE1 is up-regulated post-transcriptionally in response to energy inhibition, studies indicating that glucose metabolism is reduced in AD brains, and the observation that BACE1 protein levels are increased in post- mortem AD brains all support the hypothesis that post-transcriptional up-regulation of BACE1 in response to energy deprivation in the brain may, in part, be responsible for AP accumulation in sporadic Alzheimer's disease (SAD)- the predominant form of AD. The aim of this study is to identify the specific post- transcriptional mechanism and signaling pathway(s) leading to BACE1 up-regulation in response to energy deprivation in vitro and in vivo. Preliminary studies in 293 cells indicate that BACE1 protein increases in response to energy deprivation are mediated by the BACE1 5'-untranslated region (UTR). First, we will determine whether a similar mechanism controls BACE1 protein levels in response to energy deprivation in cultured neurons. Then, luciferase reporter assays will be used to determine whether the BACE1 5'UTR is responsible for BACE1 protein increases under glucose-deficient conditions in both 293 cells and cultured neurons. Acute pharmacological induction of metabolic stress in a transgenic mouse line lacking the BACE1 5'UTR will be used to confirm this mechanism in vivo. Preliminary experiments indicate that phosphorylation of the alpha subunit of the eukaryotic translation initiation factor 2 (elF2alpha) may be mediating post-transcriptional increases in BACE1 protein under conditions of energy deprivation. We will use 293 cells and neurons deprived of glucose in vitro, as well as brains from mice subjected to acute and chronic energy deprivation to test for activation of kinases known to phosphorylate elF2alpha. We will then determine whether pharmacological induction of elF2alpha phosphorylation through treatment with thapsigargin leads to post-transcriptional increases in BACE1 protein in vitro. Finally, to establish this pathway as potential AD drug target, we will inhibit elF2alpha phosphorylation in vitro to determine whether BACE1 protein up-regulation can be blocked under glucose-deficient conditions. The predicted increase in AD occurrence and the current lack of effective treatment options have made the development of new and more effective drugs for AD treatment a public health priority. Completion of these aims will identify novel drug targets for the development of preventative AD therapies.