Fyn modulation of APP processing

描述(申请人提供)：淀粉样前体蛋白(APP)的加工导致A？片段是阿尔茨海默病(AD)发病机制的重要组成部分。载脂蛋白E是AD发病机制中的另一个重要分子，尽管它在中枢神经系统中的功能更是鲜为人知。最近，我们发现APP和apoE受体胞质结构域结合的接头蛋白Dab1影响APP的加工和A？载脂蛋白E受体ApoER2的水平和处理。Dab1还与Fyn激酶相关，在携带Fyn突变的小鼠中发现Dab1的上调。Fyn是一种具有多种生物学功能的Src家族酪氨酸激酶，它能够磷酸化多种细胞内信号分子以响应细胞外刺激。最值得注意的是，Fyn参与了涉及学习和记忆的神经元中的信号转导过程，可能是通过对NMDA受体亚型的影响。在对这项提议的初步研究中，我们发现改变FYN水平或活动会导致应用程序交易量、应用程序稳定性和A？级别。我们还发现，Fyn对ApoER2也有类似的影响。我们假设APP的Fyn磷酸化减少了其淀粉样蛋白的形成过程。我们将测试Fyn和Dab1是协同工作，还是相互竞争，以改变APP和ApoER2的处理。最后，我们假设Fyn的生理激活促进了这些对APP和ApoER2的影响，并且对它们的调控是重要的。在Aim1中，我们将检测Fyn激酶在APP和ApoER2蛋白降解中的作用机制。在目标1A中，我们将确定APP、ApoER2和Dab1的磷酸化是否影响它们的关联和处理。在Aim 1B中，我们将确定在体内外Fyn基因敲除细胞中，或者在用siRNA还原Fyn后，APP和ApoER2的磷酸化和加工是否发生改变。在目标2中，我们将测试Fyn激活对神经元的影响。我们最初将重点研究Fyn对APP和ApoER2向细胞表面运输的影响，并确定这些影响是否调节Fyn对NMDA受体运输的影响。其次(目标2B)，我们将使用reelin作为Fyn的生理激活剂，以确定Fyn的激活如何影响神经元运输和APP和ApoER2的处理。通过这些实验，我们将测试Fyn如何调控APP的贩运、稳定性和切割，从而影响A？级别。

DESCRIPTION (provided by applicant): Processing of the amyloid precursor protein (APP) leading to the accumulation of the A? fragment, is a critical component of the pathogenesis of Alzheimer's Disease (AD). ApoE is another important molecule in AD pathogenesis, although its functions in the CNS are even less well understood. Recently, we found that Dab1, an adaptor protein that binds the cytoplasmic domains of APP and apoE receptors, affects APP processing and A? levels, and processing of the apoE receptor, ApoEr2. Dab1 also associates with Fyn kinase, and upregulation of Dab1 is found in mice carrying mutations in Fyn. Fyn is a Src family tyrosine kinase with diverse biological functions due to its ability to phosphorylate a wide variety of intracellular signaling molecules in response to extracellular stimuli. Most notably, Fyn is involved in modulating signal transduction processes in neurons involved in learning and memory, perhaps through effects on NMDA receptor subtypes. In preliminary studies for this proposal, we have found that altering Fyn levels or activity causes changes in APP trafficking, APP stability, and increased A? levels. We also found that Fyn had similar effects on ApoEr2. We hypothesize that Fyn phosphorylation of APP decreases its amyloidogenic processing. We will test whether Fyn and Dab1 work in conjunction or compete with one other to alter processing of APP and ApoEr2. Finally, we hypothesize that physiological activation of Fyn promotes these effects on APP and ApoEr2 and is important for their regulation. In Aim1, we will test the mechanism of action of Fyn kinase on APP and ApoEr2 proteolysis. In Aim 1A, we will determine whether phosphorylation of APP, ApoEr2, and Dab1 affect their associations and the processing. In Aim 1B, we will determine whether phosphorylation and processing of APP and ApoEr2 are altered in Fyn knock-out cells, in vitro and in vivo, or after reduction of Fyn with siRNA. In Aim 2, we will test the effects of Fyn activation in neurons. We will initially (Aim 2A) focus on the effects of Fyn on the trafficking of APP and ApoEr2 to the cell surface, and define whether these effects modulate the effect of Fyn on NMDA receptor trafficking. Secondly (Aim 2B), we will use reelin as a physiological activator of Fyn to define how activation of Fyn affects neuronal trafficking and processing of APP and ApoEr2. Through these experiments, we will test how Fyn regulates the trafficking, stability and cleavage of APP, thus affecting A? levels.