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

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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.