682 Tyr in the Intracellular Domain of APP Regulates Amyloidogenic APP Processing In Vivo Alessia P. M. Barbagallo1., Richard Weldon1., Robert Tamayev1., Dawang Zhou1, Luca Giliberto3, Oded Foreman2, Luciano D’Adamio1* 1 Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, United States of America, 2 Department of Laboratory Animal Health, The Jackson Laboratory, Bar Harbor, Maine, United States of America, 3 The Litwin-Zucker Research Center for the Study of Alzheimer’s Disease, The Feinstein Institute for Medical Research, North Shore – LIJ, Manhasset, New York, United States of America Abstract Background: The pathogenesis of Alzheimer’s disease is attributed to misfolding of Amyloid-b (Ab) peptides. Ab is generated during amyloidogenic processing of Ab-precursor protein (APP). Another characteristic of the AD brain is increased phosphorylation of APP amino acid Tyr682. Tyr682 is part of the Y682ENPTY687 motif, a docking site for interaction with cytosolic proteins that regulate APP metabolism and signaling. For example, normal Ab generation and secretion are dependent upon Tyr682 in vitro. However, physiological functions of Tyr682 are unknown. Methodology/Principal Findings: To this end, we have generated an APP Y682G knock-in (KI) mouse to help dissect the role of APP Tyr682in vivo. We have analyzed proteolytic products from both the amyloidogenic and non-amyloidogenic processing of APP and measure a profound shift towards non-amyloidogenic processing in APP KI mice. In addition, we demonstrate the essential nature of amino acid Tyr682 for the APP/Fe65 interaction in vivo. Conclusions/Significance: Together, these observations point to an essential role of APP intracellular domain for normal APP processing and function in vivo, and provide rationale for further studies into physiological functions associated with this important phosphorylation site. Citation: Barbagallo APM, Weldon R, Tamayev R, Zhou D, Giliberto L, et al. (2010) Tyr682 in the Intracellular Domain of APP Regulates Amyloidogenic APP Processing In Vivo. PLoS ONE 5(11): e15503. doi:10.1371/journal.pone.0015503 Editor: Maria A. Deli, Biological Research Center of the Hungarian Academy of Sciences, Institute of Biophysics, Hungary Received July 30, 2010; Accepted October 8, 2010; Published November 16, 2010 Copyright: 2010 Barbagallo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Alzheimer’s Association IIRG-09-129984 grant (LD), NIH Grants R01AG033007 (LD) and Training Program in Cellular and Molecular Biology and Genetics T32 GM007491 (RT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: luciano.dadamio@einstein.yu.edu . These authors contributed equally to this work. Introduction The most common form of dementia in the world is Alzheimer’s disease (AD), affecting about 1% of the human population by aged 65, and rising to 35–40% after age 85. Evidence points to a key role for misfolded amyloidogenic Ab peptides in the pathogenesis of AD (amyloid cascade hypothesis). The accumulation of Ab as plaques in the hippocampus and other brain regions is a key characteristic of AD pathology [1,2]. Ab peptides are generated during amyloidogenic processing of Ab-precursor protein (APP). When APP is cleaved by b-secretase, the soluble ectodomain (sAPPb) is released extracellularly whilst the 99 amino acid C-terminal fragment (C99) remains membrane bound. In a second proteolytic event, C99 is cleaved by the c- secretase. Two peptides are released, Ab peptide consisting of either 40 or 42 amino acids (Ab40 and Ab42, respectively) and an intracellular product (AID or AICD), which regulates apoptosis [3] and transcription [4]. An alternative, non-amyloidogenic pathway also exists. In this pathway APP is cleaved by a-secretase in the Ab sequence producing the soluble ectodomain (sAPPa) and the membrane bound 83 amino acid C-terminal fragment (C83). C83 is also further cleaved by the c-secretase into the P3 and AID peptides. The degree to which APP function plays a role in the pathogenesis of AD is unclear but changes in the apoptotic and axonal activities of APP may underlie some aspects of AD pathology [5,6]. However, until the in vivo functions of APP are better understood this will remain a crucial question. APP mice have retarded neuron development, reduced hippocampal neuron viability, diminished grip strength, locomotor activity and postnatal growth [7], but can be normalized by over expression of the sAPP-a ectodomain [8]. However, since the essential functions of APP are compensated for by homologues APLP1 and APLP2, the physiological significance of the short intracellular C-terminal domain remains relatively unexplored in vivo. The ,50 amino acids long APP intracellular region contains seven residues that can be phosphorylated andseveral of these amino acids are hyperphosphorylated in human AD brain. However,it remains unclear whether this is a cause or a consequence of neurodegeneration. One of these sites of particular functional significance is Tyr682, the phosphorylation of which is increased in AD patients [9], especially in vasculature tissue of the PLoS ONE | www.plosone.org 1 November 2010 | Volume 5 | Issue 11 | e15503