APP Processing and Tyrosine682 Figure 3. Normal expression of the mutant APP allele. A, The genetic manipulation of the APP gene locus in APPYG/YG mice does not affect transcription/splicing of APP since mRNA APP levels are comparable to those transcribed in age-matched APPWT/WT mice (analogous data were obtained using Beta Actin as housekeeping gene). B, APP and APP-derived CTFs are processed by the lysosomes. Inhibition of lysosomal activity by chloroquine (Chl.) results in accumulation of APP and APP derived fragments. The asterisks indicate APP-CTFs that are derived by cleavages in the ectodomain NH2-terminal to the site processed by BACE1. The effectiveness of Chl. in inhibiting lysosomal degradation (inhibition occurs at 50 mM concentration but not at 5 mM) is confirmed by the accumulation on LC3II [78]. The antibody against LC3 is from Cell Signaling. doi:10.1371/journal.pone.0015503.g003 not always reproducible (APP levels are similar between the two strains in the experiments shown in Fig 2C, Fig. 4 and Fig. 5). The reason for this variability is not presently understood. Since a- and b-cleavages are mutually exclusive, an increase in one should be compensated by roughly equal decrease in the other if all full length APP is cleaved, in similar proportions, by either a- or b-secretase. However, we report a ,15-fold increase in soluble sAPP-a but only a ,3.5 -fold decrease in sAPP-b. Unless in vivo sAPP-b is cleared more efficiently than sAPP-a, these differences are not consistent with the aforementioned model. A recent report shows that APP is cleaved in the ectodomain buy an alternative, albeit yet to be identified, protease [21]. In addition, a large fraction of full length APP is processed by lysosomes, presumably after APP is internalized [22] (Fig. 3B). The YG mutation could reduce BACE and lysosomal degradation of APP, if mutant APP has impaired endocytosis [both processes are largely dependent of APP endocytosis [22,23,24]). This would also explain the vast increase in a-secretase processing of the YG APP mutant. This hypothesis is presently being investigated. Also C83 and C99 undergo lysosomal degradation (Fig. 3B), indicating that these APP metabolites are not exclusively cleaved by g-secretase. Interestingly, inhibition of lysosomal degradation results in the appearance of several COOH-terminal APP fragments larger than C99 [see asterisks in Fig. 3B for both primary neurons and primary mouse dermal fibroblasts (MDFs)]. These fragments are either intermediated of APP degradation in lysosomes, or are produced by processing of APP in regions NH2- terminal to the BACE1 cutting site, suggesting that the APP ectodomain can be processed by several unknown proteases, in addition to a- and b-secretase. The decrease in Ab40 level (25%) is not as pronounced as the reduction in sAPPb levels. This apparent discrepancy can be explained by either reduced clearance of brain Ab, a compensa- tory increase in c-cleavage of C99, or by reduced clearance of C99 by the lysosomes. These possibilities are being investigated. As noted above, intracellular transport and localization of APP are critical components of APP processing and Ab production. In fact, a-secretase cleaves mAPP en route to or on the plasma membrane. b-secretase predominantly cleaves mAPP in early endosomes [23,24] while C99 and C83 are processed by the c- secretase in endocytic compartments [24]. Thus, the shift toward the non-amyloidogenic processing in APPYG/YG mice may involve a role of Tyr682 in trafficking of APP along the secretory pathway. However, the YG mutation neither altered the imAPP/mAPP ratio in brains (Fig. 4A and B) and primary mouse dermal fibroblasts (MDFs) (Fig. 4C), nor changed the levels of plasma membrane mAPP in MDFs (Fig. 3C). Normal brain organization and distributions of neural proteins in APPYG/YG mice A number of studies suggest important neurological roles for APP and APP polypeptides derived by secretases processing. sAPP-a is neuro-protective [25,26,27,28]. A metabolite of sAPP-b interacts with DR6 to trigger neuronal death [29]. Ab is critical for LTP induction and memory acquisition [30]. AID/AICD modulates cell death, gene transcription and Ca++ homeostasis [3,4,31,32,33,34,35,36,37,38,39,40]. Because APP derived poly- peptides are significantly changed in APPYG/YG mice, we analyzed whether these mutant mice show abnormalities in brain organization. First, a general histopathological examination of the APPYG/YG mouse showed the following. All mice had minimal multifocal myofiber degeneration affecting the appendicular musculature, primarily, the biceps femoris, quadriceps, and triceps brachii muscles. Randomly scattered throughout the striated muscles were small clusters of swollen muscle fibers with increased cytoplasmic eosinophilia and occasional karyorrhexis (Figure 5). These myofibers were surrounded by myocytes with variable cross sectional diameter and occasional rowing of central nuclei interpreted as myofiber regeneration. PLoS ONE | www.plosone.org 6 November 2010 | Volume 5 | Issue 11 | e15503