Figure 6. Coronal sections comparing brain anatomy of APPwt/wt with APPYG/YG mice. A–E coronal sections of APPwt/wt mouse, F–J coronal sections of APPYG/YGmouse. A, F - Olfactory bulbs. B, G- Cerebral cortex. C, H- Thalamus. D, I- Midbrain. E, J- Medulla. Ob- olfactory bulb, r-retina, cc- cerebral cortex, s- striatum, hi- hippocampus, th- thalamus, mb- midbrain, ce- cerebellum. doi:10.1371/journal.pone.0015503.g006 Thr668 [67,72]. These data suggest that phosphorylation–dephos- phorylation on Tyr682 and Thr668 modulates APP interactions and function. Notably, Tyr682 and Thr668 phosphorylation is increased in AD brains [9,73]. The APP/Fe65 interaction is the best characterized of many potential APP/APP C-terminal binding protein complexes. In vitro studies have suggested a role for APP/ Fe65 complexes in APP metabolism, and for AID-AICD/Fe65 in gene transcription [4,62,74]. Tyr682 is essential for a robust APP/ Fe65 interaction to occur in vitro [75] and, as noted above, other in vitro evidence shows that Tyr682phosphorylation abolishes docking of Fe65 to APP. To establish whether this important role of Tyr682 is true in vivo co-IP of APP and Fe65 was carried out using brain tissue from APPYG/YG, APP2/2 and APPwt/wt control mice APP Processing and Tyrosine682 (Fig. 8). Panel A shows co-IP using a-APP antibody. The loss of detectable signal for Fe65 in both APP2/2 and APPYG/YG mice compared to APPwt/wt control clearly demonstrates that Tyr682 is specifically required for this interaction to occur. Conversely Panel B shows an IP experiment using a-Fe65 antibody and clearly shows an absence of binding of APP in APPYG/YG mice, again demonstrating the necessity of Tyr682 in the APP/Fe65 interaction. Discussion Mis-folding of amyloidogenic Ab peptides, particularly Ab42, is a key feature of the AD pathology. APP Y682G mutation in mice clearly results in a large redistribution of APP towards non- amyloidogenic pathway; sAPP-a and C83 are greatly increased while sAPP-b and Ab40 are decreased (Fig. 2), thus demonstrating the necessary role of the C-terminal in normal activity of the amyloidogenic pathway in the brain and consistent with the results observed in vitro. It is not yet apparent why a concurrent reduction in C99 or p-C99 was not observed. It is interesting to speculate, based upon previous findings, how this profound shift in APP processing may influence physiology in APPYG/YG mice. sAPP-a for instance is a proposed growth factor with neuroprotective properties, therefore the 15-fold over expression may result in different growth characteristics and a resistance to stress, although no differences in brain organization were apparent in our analysis (Fig. 5, 6). A possible mechanism to explain this shift toward the non- amyloidogenic processing may involve the essential role of Tyr682 for normal endocytosis of APP as previously shown in vitro [11]. A fraction of APP is cleaved by a-secretase in a post-Golgi compartment or at the plasma membrane. Alternatively, some APP is processed by b-secretase in the Golgi or in late endosomes following internalization from the cell membrane. In addition Ab levels tightly correlate with APP internalization such that Ab secretion is significantly decreased when APP endocytosis is compromised in vitro [23,24]. However our initial analysis of APP localization shows no difference in cell surface APP (Fig. 4C). We also demonstrate that Tyr682 is necessary for interaction between APP and binding partner Fe65 in vivo (Fig. 8). Interestingly Fe65 also plays role in endocytosis of APP. Fe65 simultaneously binds to the cytoplasmic tail of APP and of LRP1 into a trimeric complex [62,76,77]. This interaction results in accelerated endocytosis of APP via clathrin-coated pits and in delivery to late endosomal compartments for cleavage by b- and c- secretase to generate Ab [72]. The decrease in amyloidogenic processing in APPYG/YG mice may be consistent with this data. Another known function of the APP/Fe65 interaction, which should be absent in APPYG/YG mice, is the transcriptional activity of AID/Fe65/Tip60 complex [4], although further investigation will be needed to determine if this is true. Also of note is that phosphorylation of Tyr682 also disrupts interaction of APP with Fe65 and other PTB domain proteins [13,14] and this may be one commonality between APPYG/YG mice and AD brain. A comparative analysis between APPYG/YG, APPTA/TA and control mice clearly indicates steady-state APP phosphorylation predominantly occurs on Thr668 and not Tyr682 in normal brain (Fig. 2D). Counter intuitively, this observation may exemplify the importance of Tyr682 phosphorylation. Evidence shows Tyr682 is hyperphosphorylated in the AD Brain [9,10]. It is plausible that excessive phosphorylation at this functionally important residue could lead to toxic effects, given its relative scarcity under normal conditions. For example, if Tyr682 phosphorylation plays a role in targeting APP for degradation via secretase, lysosomal or PLoS ONE | www.plosone.org 8 November 2010 | Volume 5 | Issue 11 | e15503