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

早老素（PS）在asas的产生中起着核心作用，因为功能突变的丧失和基因缺失导致asas产生的急剧减少。在最初的资助期间，我们可以证明很大一部分PS是针对细胞表面的，并且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向总理贩运PS。最近描述的两种蛋白Nicastrin和Aph-1与PS相互作用，我们希望利用gfp标记的蛋白确定它们在PS运输中的作用，反之亦然。我们还计划定量分析APP从ER向高尔基体的转运。为此，我们将VSVG的热敏结构域与APP-YFP的跨膜和胞质结构域融合。该结构允许使用视频显微镜定量测量APP从ER到高尔基体的输出速率。我们将分析PS、Aph-1和Nicastrin对这一转运步骤的影响。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，我们想要表征BACE的运输，并找出它与底物APP的结合点。我们想通过在海马神经元中表达gfp标记的PS和Nicastrin来扩展这些研究，并分析它们的运输。

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.