Project Summary Our preliminary studies show that the APP E590D variant, identified in two AD patients’ brains, substantially induces more Aβ than APP-WT in vitro cell line and primary mouse cortical neurons. This APP E590D variant also significantly increases APP protein stability and APP protein's endocytic process more than APP-WT. Compared to APP-WT and many other APP mutants, APP E590D generates not only Aβ peptide but also another extracellular 15kda fragment. Therefore, we take advantage of the human neurons directly converted from primary fibroblasts to test the hypothesis that APP E590D promotes Aβ pathology while inducing abnormal endocytosis of APP protein, mitochondrial dysfunction, and impairment of synaptic integrity in human neurons. Unlike most other classified pathogenic APP missense variants located in the Aβ sequence or C terminus, the APP E590D variant site is six residues away from the β-cleavage site beyond the Aβ sequence at the N terminus APP protein, which results in a very conservative protein structure. Considering the patient carrying the APP E590D variant has not only excessive amyloid deposition but also shows other non-AD-associated neuropsychological dysfunctions at a very young age, we will (1) identify new C terminal cleavage site of APP beyond the β-secretase cleavage using a mass spectrometry approach; (2) generate Aβ sequence-humanized APP E590D knock-in mouse model of AD and test the pathogenic role of APP E590D in vivo. The successful conclusion of these studies will (1) determine the pathogenic role of APP E590D in human neurons and mice brains in vivo and uncover a new APP cleavage site with its unique fragment from APP E590D; (2) provide a new APP E590D-based knock-in animal model of AD for other investigators in the field.