These data suggest a presence of a potential compensatory mechanism that maintains the total level of sAPP in the brain regardless of ADAM10 expression levels. We also measured the levels of secreted APP (sAPPα, sAPPβ,

and total sAPP) in cerebrospinal fluid (CSF) and found similar patterns depending on the ADAM10 genotypes (Figure S2A). Levels of endogenous Aβ40 and Aβ42 (measured by ELISA) were significantly lower in ADAM10-WT mice compared to the nontransgenic controls, while the levels were dramatically higher in ADAM10-DN mice (Figure S2B). For mice expressing either Q170H or R181G mutations, a trend was observed in which endogenous Aβ levels were increased compared to ADAM10-WT mice. Taken together, these data reveal that both ADAM10 LOAD prodomain

mutations attenuate but do not entirely abolish α-secretase activity of ADAM10 on www.selleckchem.com/products/Y-27632.html APP. Next, we asked selleck screening library whether the prodomain mutations affect the cleavage of other ADAM10 substrates besides APP (Pruessmeyer and Ludwig, 2009). We examined the processing of candidate ADAM10 substrates in the brain including APLP2, Notch1, and N-cadherin. The processing of APLP2 showed very similar patterns to those of APP in that ADAM10-WT overexpression resulted in the increased cleavage of mature APLP2, while this was attenuated in mice expressing either of the two LOAD mutations (Figure S2C). Interestingly, ∼57-kDa-sized C terminus-truncated soluble APLP2, analogous to

the soluble APP cleavage product, was detected more abundantly in ADAM10-WT than the LOAD mutant mouse brains. In contrast, western blot analysis of adult brains and primary neurons derived from ADAM10 transgenic mice showed ADAM10 overexpression barely affected N-cadherin level or N-cadherin-CTF generation (Figure S2D). In contrast to embryonic brains, most nearly of Notch1 protein in adult brains is cleaved and present as extracellular truncated forms in the membrane. We found that neither WT nor mutant ADAM10 overexpression changed the level of full-length or truncated forms of Notch1 (Figure S2E). Together, these data suggest that the processing of APP family proteins is particularly vulnerable to loss-of-function mutations in ADAM10. To evaluate the effects of the two LOAD ADAM10 mutations on AD pathogenesis, we crossed transgenic mice expressing WT or mutant forms of ADAM10 with Tg2576 AD mice and assessed APP processing, Aβ levels, and amyloid plaque load. As in the ADAM10 transgenic mice, the prion protein promoter was utilized in the Tg2576 mice to express human APPswe. Western blot analysis of mouse brain lysates revealed that APP expression was several-fold higher in Tg2576 mice than in nontransgenic control. However, ADAM10 expression in Tg2576/ADAM10 double-transgenic mice was maintained at similar levels to those in parental ADAM10 single-transgenic mice (Figures 2A and S3A).