The cellular basis of APP processing: trafficking and assembly of gamma-secretases and its substrates

早老素（PS）在Ablation产生中起核心作用，因为功能缺失突变和基因缺失导致Ablation产生的显著减少。在最初的资助期间，我们可以证明，PS的一个显着的部分是针对细胞表面，PS参与APP的再内化，从而直接影响其底物的运输和加工。我们现在想研究如何监管PS向PM的贩运。两个最近描述的蛋白质，Nicastrin和Aph-1，与PS相互作用，我们想确定他们的作用，在运输PS，反之亦然，使用GFP标记的蛋白质。我们还计划定量分析APP从ER到高尔基体的运输。为此，我们将VSVG的热敏结构域融合到APP-YFP的跨膜和胞质结构域。这种结构允许使用视频显微镜定量测量APP从ER输出到高尔基体的速率。将分析PS、Aph-1和Nicastrin对该转运步骤的影响。最后，我们想表征APP及其加工酶在海马神经元中的转运。在这里，我们想描述BACE的运输，并找出它满足其基板，APP。我们希望通过表达GFP标记的PS和Nicastrin在海马神经元和分析他们的运输扩展这些研究。

Presenilins (PS) play a central role in Aß production since loss of function mutations and gene deletions result in a dramatic reduction of Aß generation. During the initial funding period we could demonstrate that a significant fraction of PS is targeted to the cell surface and that PS are involved in reinternalization of APP, thus directly influencing trafficking and processing of their substrates. We now want to study how PS trafficking to the PM is regulated. Two recently described proteins, Nicastrin and Aph-1, interact with PS and we want to determine their role in trafficking of PS and vice versa using GFP-tagged proteins. We also plan to quantitatively analyze the transport of APP from the ER to the Golgi. To this end we fused a heat sensitive domain of VSVG to the transmembrane and cytoplasmic domain of APP-YFP. This construct allows to quantitatively measure the rate of APP export from the ER to the Golgi using video microscopy. The influence of PS, Aph-1 and Nicastrin on this transport step will be analyzed. Finally we want to characterize the transport of APP and its processing enzymes in hippocampal neurons. Here we want to characterize the transport of BACE and find out where it meets its substrate, APP. We want to extend these studies by expressing GFP-tagged PS and Nicastrin in hippocampal neurons and analyze their transport.