Compared to neurons expressing APP only, mature APP levels were significantly diminished in those expressing APP plus BACE1-WT or APP plus BACE1-CA4, suggesting that a significant proportion of APP is cleaved by BACE1 (Fig. 5d). Consistently, neurons KPT-330 datasheet coexpressing APP and either BACE1-WT or BACE1-CA4 secreted ~6.5-fold higher amounts of Aβ40 and ~2.5-fold or ~2.9-fold higher amounts of Aβ42, respectively, than those expressing APP alone (Fig. 5e and f). Together, BACE1-WT and BACE1-CA4 exerted similar Aβ-promoting effects, suggesting that β-cleavage of APP does not depend on raft localization of BACE1. β-CTF is predominantly

localized in nonraft Inhibitors,research,lifescience,medical membrane domains To evaluate the β-cleavage of APP in raft and nonraft domains, we performed Western blot analysis of APP CTF. Western Inhibitors,research,lifescience,medical blots of RIPA lysates revealed that levels of β-CTF and β′-CTF (derived from alternative BACE1 cleavage of APP between Tyr10 and Glu11 within the Aβ region) were remarkably increased and those of α-CTF (derived from α-secretase cleavage of APP Inhibitors,research,lifescience,medical between Lys16 and Leu17 within the Aβ region) decreased in neurons expressing APP plus BACE1-WT or APP plus BACE1-CA4, compared to those expressing APP alone (Fig. 6a). Subsequently, we examined the distribution of APP CTF in raft and nonraft fractions following sucrose density gradient fractionation. Immunoprecipitation–Western blot analysis revealed

that the majority of β-CTF and β′-CTF was recovered in nonraft fractions (fractions 8–10) of neurons expressing APP plus BACE1-WT or APP plus BACE1-CA4, whereas only low levels were present in the raft fraction (fraction 4). No differences in the localization pattern of CTFs were observed between neurons expressing BACE1-WT and BACE1-CA4 Inhibitors,research,lifescience,medical (Fig. 6b). Figure 6 Predominant localization of APP CTF in nonraft domains of neurons coexpressing APP and BACE1. (a) RIPA or Inhibitors,research,lifescience,medical CHAPS extracts

of neurons coexpressing APP and either mock or BACE1-WT or BACE1-CA4 were subjected to Tris/Tricine SDS-PAGE and immunoblotting with … Next, we evaluated the β-cleavage of APP by endogenous BACE1. For this purpose, primary neurons overexpressing Swedish mutant APP, a preferred substrate Ribonucleotide reductase of BACE1, via recombinant adenoviruses were treated with a γ-secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-(S)-phenylglycine t-butyl ester) (Dovey et al. 2001) that augments the levels of APP CTF. The distributions of endogenous BACE1 and APP CTF were then analyzed following sucrose density gradient fractionation. Bands of BACE1 were observed in both raft and nonraft fractions and faint bands probably representing dimeric BACE1 were additionally detected in nonraft fractions (Fig. 6c). Higher levels of β-CTF and β′-CTF were obviously recovered in nonraft fractions than in the raft fraction (Fig. 6d). These results suggest that β-cleavage of APP by overexpressed as well as endogenous BACE1 occurs mainly in nonraft fractions.