Our subsequent analysis revealed that BACE1-WT and BACE1-CA4 promoted neuronal production of Aβ40 and Aβ42 from endogenous and overexpressed APP to similar extents. These results clearly indicate that BACE1 raft localization does not affect Aβ production in neurons, in agreement with a previous report (Vetrivel et al. 2009) in which BACE1-null fibroblasts and mouse N2a neuroblastoma cells were used for experiments. Furthermore, we observed that both β-CTF and β′-CTF metabolites derived from BACE1 processing were mainly localized in the nonraft fractions of neurons expressing APP and either BACE1-WT or BACE1-CA4. We additionally observed that β-CTF derived from APP cleavage Inhibitors,research,lifescience,medical by endogenous BACE1

also distributes mainly in nonraft fractions of neurons expressing Swedish

mutant APP. These data support the view that BACE1 cleavage of APP occurs mainly in the nonraft domains in neurons. A mutant form of BACE1 (BACE1-GPI) in which transmembrane and cytoplasmic domains are replaced with glycosylphosphatidylinositol Inhibitors,research,lifescience,medical (GPI) anchor attachment signal that preferentially localizes in lipid rafts was reported to exhibit increased β-cleavage activity (Cordy et al. 2003). However, a recent study has revealed that the increase in Aβ secretion under the Inhibitors,research,lifescience,medical condition of BACE1-GPI expression is mainly due to reduced APP cleavage at the β′-site, compared with wild-type BACE1 (Vetrivel et al. 2011). It Inhibitors,research,lifescience,medical is likely that β-site cleavage efficiency of BACE1 is essentially unaltered by its association with lipid rafts. Based on the present and earlier results, we propose a dynamic model of neuronal Aβ generation,

as illustrated in Fig. 9. First, APP is cleaved by BACE1 mainly in nonraft domains, generating β-CTF. Next, β-CTF mobilizes from nonraft to raft domains through unknown mechanisms, where it is finally cleaved by γBIBW2992 in vivo -secretase, resulting in Aβ production. Thus, identification Inhibitors,research,lifescience,medical of the specific factors involved in the transport of β-CTF from nonraft to raft domains is an important subject of focus for future research. Figure 9 Hypothetical model for APP processing. APP processing in neurons is possibly a dynamic process whatever involving three steps. (I) APP is cleaved by BACE1 mainly in nonraft domains, generating β-CTF. (II) β-CTF is transported from nonraft to raft … Endosomes and the trans-Golgi network are important organelles for the production of Aβ and β-CTF. A previous study (Vetrivel et al. 2009) presented evidence that palmitoylation of BACE1 does not regulate its distribution in these organelles or at the cell surface. Thus, BACE1-WT and BACE1-CA4 likely exert β-cleavage activity in nonraft domains of the same subcellular sites. Interestingly, the β′-CTF level was higher than that of β-CTF in neurons coexpressing APP and BACE1. Consistently, a recent study has indicated that β′-cleavage is a major processing event that occurs with human APP in neuronal cultures (Zhou et al.