The impact of the mutations on Aβ accumulation and plaque deposition was further assessed by crossing these ADAM10 transgenic mice with Tg2576 mice, a well-characterized AD mouse model that overexpresses human APP Swedish mutation (APPswe) and is known to cause an APP ectodomain

cleavage shift that favors the β- over the α-site. We also assessed the effect of the LOAD mutations on adult hippocampal neurogenesis and, finally, we explored the underlying molecular R428 molecular weight mechanisms by which the LOAD mutations in the ADAM10 prodomain attenuate α-secretase activity. To test the in vivo effects of the two LOAD-associated ADAM10 mutations on α-secretase activity, we generated transgenic mice overexpressing human WT ADAM10, ADAM10 harboring the LOAD-associated mutations Q170H or R181G, and an artificial dominant-negative (DN) mutation, E384A. All transgenes were driven by the mouse prion protein promoter (MoPrP) and tagged with hemagglutinin (HA) at the C terminus of the protein (Figures S1A and S1B available online). For each ADAM10 genotype, we obtained three WT, three Q170H, eight R181G, and three DN F1 transgenic mice, which were bred with nontransgenic littermates to maintain mouse lines. The brains from F2 and F3 progenies of each line were analyzed for ADAM10 expression and APP processing. Western blot analysis of 12-week-old

mouse brains revealed INCB024360 that a WT transgenic line (WT-58) expressed ∼2.5-fold higher level of mature ADAM10 in brain than nontransgenic control (Figure S1C). In addition to the pro and mature forms of ADAM10, high levels of ADAM10-CTF (∼10 kDa) were detected in the

membrane fraction (Figures 1A–1C and S1D). Previous studies have shown that these ADAM10-CTFs are generated by ectodomain shedding of ADAM10 mature forms (Parkin and Harris, 2009 and Tousseyn et al., 2009). Interestingly, as compared to the ADAM10-WT transgenic mice, the levels of ADAM10-CTF were significantly reduced in mice expressing either of the two LOAD mutations and were undetectable in mice expressing DN mutation (Figures 1A and 1C). This pattern of reduced ADAM10-CTF was consistently observed Dipeptidyl peptidase in all the ADAM10 LOAD and DN mutant lines as compared to WT transgenic lines (two WT, three Q170H, six R181G, and three DN ADAM10 transgenic lines) and in the three single F1 mice (one WT and two R181G, which failed to produce progeny). The decrease in ADAM10-CTF generation was also detected in primary cortical neurons derived from the LOAD mutant mice (Figure S1E). These results indicate that both the LOAD and DN ADAM10 mutations decreased ectodomain shedding of the metalloprotease. Consistent with our findings, previous studies have shown that artificial mutations at the prodomain cleavage or catalytic sites, which block enzyme activity of the corresponding ADAM proteases (ADAM13 and ADAM19), also result in the prevention of its own ectodomain shedding at their cysteine-rich domains (Gaultier et al., 2002 and Kang et al.