Figure 4. Maturation and membrane levels of APP are unaffected by the YG mutation. A, Immunoblot analysis comparing imAPP and mAPP between APPwt/wt and APPYG/YG mice (n = 3). B, Quantitative analysis of panel A shows no differences in mAPP and imAPP levels between the two genotypes. C, Biotynilation experiment in APPwt/wt and APPYG/YG MDFs shows comparable levels of im and mAPP, as well as cell membrane levels of mAPP. doi:10.1371/journal.pone.0015503.g004 APP Processing and Tyrosine682 Coronal step sections of the brain were made at the five following levels- olfactory bulbs, cerebral cortex, thalamus, midbrain and medulla. Each of these regions was further serially sectioned at 250-micron intervals and stained with H&E. Histological examination revealed no structural or anatomical differences between the APPYG/YG and the wild type mice (Fig. 6). Next, we tested distribution of neural proteins using a biochemical approach. Recent evidence suggest a role for APP in synaptic function and numerous data support a role for synaptic dysfunction underlying subtle memory changes in AD [41]. Since the presynaptic regions of neurons are thought to be the main source of Ab in the brain, attention has been focused on axonal APP trafficking. These studies have unveiled an active role for APP in axonal transport. APP is transported anterogradely by conventional kinesin in tubular vesicles [42,43,44,45,46,47,48]. Although a direct interaction of APP with the motor protein kinesin-1 has been proposed [49], following studies have contradicted this conclusion [50,51] and shown that APP interacts with kinesin-1 trough the APP-interacting proteins JIP1, a c-Jun N-terminal kinase JNK-signaling scaffold protein [51,52,53]. It has also been proposed that b- (BACE1) and c-secretases transported in APP-containing vesicles and that APP functions as a receptor for the cargo transport [54]. A number of observations suggest that microtubule-dependent axonal transport is impaired in AD human brains [5,55,56,57,58] as well as APP transgenic mice [5,59,60]. Because of the indication that the intracellular domain of APP is important for axonal transport of APP and cargo molecules, we analyzed the synaptic distribution of neural proteins. We studied APP. BACE1, a component of the c- secretase complex, Nicastrin, and synaptic proteins/receptors, such as PSD95, SVP38, the Glutamate receptors NMDAR1, NMDAR2A, NMDAR2B and GLuR2/3/4. The synaptic levels of these proteins were unchanged (Fig. 7), further supporting the notion that the YG mutation does not affect APP maturation and trafficking and that APP may not be a regulator of fast anterograde axonal transport. Absence of APP/Fe65 interaction in APP Y682G mutant mice Several cytosolic proteins bind APP. These APP-interacting proteins regulate both APP processing and functions of APP polypeptides in vitro [51,52,53,61,62,63,64,65,66,67,68]. However, the in vivo relevance of these findings is still unclear. Most of these interactions involve the YENPTY sequence (amino acids 682–687) of APP. Phosphorylation of APP is consequential. Some proteins interact with APP only when Tyr682 is phosphorylated [14,69,70,71]; others, like Fe65, Fe65L1 and Fe65L2, only when this tyrosine is not phosphorylated [13]. The same is true for Figure 5. Alterations in quadriceps muscle from two APPYG/YG mice. A, normal striated muscle. B, muscle fibers undergoing regeneration. doi:10.1371/journal.pone.0015503.g005 PLoS ONE | www.plosone.org 7 November 2010 | Volume 5 | Issue 11 | e15503