APP MEDIATED SIGNALING EVENTS IN P53 INHIBITION

阿尔茨海默氏病（AD）是进行性阿尔茨海默病最常见的原因。 老年人的认知缺陷。 这种至今无法治愈的疾病 威胁到越来越多的老年人的健康， 美国的 照顾这些病人的经济成本是 巨大的。 有证据表明APP在AD中起重要作用 发病机制 然而，APP在中枢神经系统中的基本功能在很大程度上仍然存在， 晦涩难懂。 我们的长期目标是描述APP的功能 并确定是否与年龄相关的APP功能障碍 导致AD的神经退行性变。 在初步研究中，我们 发现野生型而非家族性AD（FAD）相关APP受到保护 在APP缺陷的神经母细胞瘤细胞系（B103）中抗凋亡。 APP的抗凋亡功能与其抑制细胞凋亡的能力相关。 关键促凋亡分子，肿瘤抑制因子p53。 拟议 研究，我们将剖析APP介导的抑制机制 p53与凋亡 首先，我们将确定APP是否降低p53 活性通过抑制p53 C-末端磷酸化， p53激活。神经元细胞培养物将用不同的 细胞凋亡诱导剂，以及APP对p53 C-末端的影响 磷酸化将通过蛋白质印迹分析来测量， 对p53的磷酸化C末端具有特异性的抗体。 APP的 抑制C-末端磷酸化和p53活化的能力将 在表达野生型、FAD突变体或 移码突变型APP（在散发性AD中发现的截短APP）。 接下来我们 将决定APP介导的信号事件，负责p53 抑制作用 APP可能作为细胞表面受体传递 外部信号进入细胞，这可能是p53抑制的原因。 为了验证这一假设，我们将产生嵌合APP受体， 确定嵌合APP的化学诱导的二聚化是否 受体将抑制神经元细胞中的p53活化和凋亡。 如果是这样，我们将确定APP的细胞内区域是否 受体和最近表征的与APP相互作用的蛋白质， 例如Fe 65和X11，是APP的p53抑制功能所必需的。 我们还将确定FAD相关突变是否会减少APP的受体- 介导的功能。 剖析分子 APP调节神经元细胞死亡的潜在机制， 生存将大大扩展我们对这种AD作用的理解 遗传标记在AD发病中的作用。

Alzheimer's disease (AD) is the most common cause of progressive cognitive deficits in elderly people. This as yet incurable disease threatens the health of an increasing number of the elderly in the United States. The financial costs of caring for these patients are enormous. Evidence suggests that APP plays an important role in AD pathogenesis. However, basic functions of APP in the CNS remain largely obscure. Our long term goals are to characterize the functions of APP in the CNS and to determine if age-related dysfunction of APP contributes to neurodegeneration in AD. In our preliminary studies, we found that wild-type, but not familial AD (FAD)-linked APP, protected against apoptosis in an APP-deficient neuroblastoma cell line (B103). APP's antiapoptotic function correlated with its ability to inhibit a key pro-apoptotic molecule, tumor suppress factor p53. In the proposed studies, we will dissect mechanisms underlying APP-mediated inhibition of p53 and apoptosis. First, we will determine if APP decreases p53 activity by inhibiting p53 C-terminal phosphorylation, which regulates p53 activation. Neuronal cell cultures will be challenged with different apoptosis inducers, and the effect of APP on p53 C-terminal phosphorylation will be measured by western blot analysis with antibodies specific to the phosphorylated C-terminus of p53. The APP's ability to inhibit C-terminal phosphorylation and activation of p53 will be analyzed in B103 cells expressing wild-type, FAD-mutant, or frameshift-mutant APP (a truncated APP found in sporadic AD). Next, we will determine the APP-mediated signaling events responsible for p53 inhibition. APP may function as a cell-surface receptor relaying outside signals into cells, which may be responsible for p53 inhibition. To test this hypothesis, we will generate a chimeric APP receptor and determine if chemically-induced dimerizaiton of the chimeric APP receptor will inhibit p53 activation and apoptosis in neuronal cells. If so, we will then determine if the intracellular region of the APP receptor and recently characterized proteins that interact with APP, such as Fe65 and X11, are required for APP's p53 inhibitory function. We will also determine if FAD-linked mutations diminish APP's receptor- mediated function in neuronal cultures. Dissecting the molecular mechanisms underlying APP's regulation of neuronal cell death and survival will greatly expand our understanding of the role of this AD genetic marker in the development of AD.