Fyn modulation of APP processing

描述（由申请人提供）：淀粉样前体蛋白（APP）的加工导致A？片段，是阿尔茨海默病（AD）发病机制的关键组成部分。ApoE是AD发病机制中的另一个重要分子，尽管其在中枢神经系统中的功能尚不清楚。最近，我们发现Dab1，一种结合APP和apoE受体细胞质结构域的衔接蛋白，影响APP加工和A？以及apoE受体ApoEr2的加工过程。Dab1也与Fyn激酶相关，并且在携带Fyn突变的小鼠中发现Dab1的上调。Fyn是一种Src家族酪氨酸激酶，由于其能够磷酸化多种细胞内信号分子以响应细胞外刺激，因此具有多种生物学功能。最值得注意的是，Fyn可能通过对NMDA受体亚型的影响，参与调节与学习和记忆有关的神经元的信号转导过程。在本提案的初步研究中，我们发现改变Fyn水平或活性会导致APP贩运、APP稳定性和A？的水平。我们还发现Fyn对ApoEr2也有类似的作用。我们假设APP的Fyn磷酸化会减少其淀粉样变性过程。我们将测试Fyn和Dab1是协同工作还是相互竞争来改变APP和ApoEr2的处理。最后，我们假设Fyn的生理激活促进了APP和ApoEr2的这些作用，并且对它们的调节很重要。在Aim1中，我们将测试Fyn激酶对APP和ApoEr2蛋白水解的作用机制。在Aim 1A中，我们将确定APP、ApoEr2和Dab1的磷酸化是否会影响它们的关联和加工。在Aim 1B中，我们将确定在体外和体内Fyn敲除细胞中APP和ApoEr2的磷酸化和加工是否发生改变，或者用siRNA还原Fyn后是否发生改变。在Aim 2中，我们将测试Fyn激活对神经元的影响。我们将首先（Aim 2A）关注Fyn对APP和ApoEr2向细胞表面运输的影响，并确定这些影响是否会调节Fyn对NMDA受体运输的影响。其次（Aim 2B），我们将使用reelin作为Fyn的生理激活剂来定义Fyn的激活如何影响APP和ApoEr2的神经元运输和加工。通过这些实验，我们将测试Fyn如何调节APP的转运、稳定性和裂解，从而影响A？的水平。

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.