This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Alzheimer's disease (AD) is definitively diagnosed neuropathologically by the presence of senile plaques and neurofibrillary tangles in the limbic and association cortices. The predominant component of the extracellular senile plaque is amyloid beta (A-beta), a 39- to 43-amino acid peptide that is proteolytically derived from amyloid precursor protein (APP). The amyloid precursor protein (APP) must fulll important roles based on its sequence conservation from y to human. However, the neuronal function of this ubiquitously expressed protein has not yet been elucidated. Although the pathogenetic mechanism(s) of AD remain to be determined, genetic, histopathologic, and biochemical evidence from humans, cell lines, and animal models implicates A-beta as a key factor in the neurodegenerative process. Therefore, one of the most prominent approaches in therapeutic development is to decrease A-beta production by inhibiting the secretases that release this peptide from APP.
The aim of this project is to extend characterization of APP, to elucidate its normal role(s) and to understand how inhibiting the secretases may affect its physiologic function(s). Candidate binding partners have been crosslinked to full-length, plasma membrane APP stably expressed by human embryonic kidney 293 cells (HEK293) expressing APP751 (HEK275) or rat embryonic day 18 primary neurons infected with a virus expressing APP. Notch2 was identied as a potential APP binding partner based on mass spectrometry analysis of APP complexes immunopuried from neurons. To conrm the interaction between Notch2 and APP, we carried out immunoprecipitation studies in HEK275 cells transiently expressing full-length Notch2 using Notch2 antibodies. The results indicated that APP and Notch2 interact in mammalian cells, and conrmed our initial ndings. Interestingly, Notch1 also coimmunoprecipitated with APP, suggesting that APP and Notch family members may engage in intermolecular crosstalk to modulate cell function. Cotransfection of APP/CFP and Notch2/YFP into COS cells revealed that these two proteins colocalize on the plasma membrane. Intracellularly, however, although some APP and Notch molecules colocalize, others reside in distinct locations. The discovery of proteins that interact with APP may aid in the identication of new functions for APP. A manuscript describing these results was recently published in the Journal of Neuroscience Research.
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