However, immunoprecipitation followed by WB analysis using a combination of antibodies targeting the ADAM10 prodomain and cytosolic domain revealed that the

cleaved ADAM10 prodomain is undetectable (Figures 7A–7D). These results suggest that the liberated ADAM10 LY294002 molecular weight prodomain is rapidly degraded following cleavage in brain and that the impact of the liberated prodomain on ADAM10 enzyme activity is likely to be minimal. This also implies that the ADAM10 LOAD mutations may affect the prodomain function prior to its liberation. Next, we asked whether the prodomain mutations interfere with the cellular trafficking of ADAM10. Previously, it has been shown that the introduction of an artificial mutation (Leu73Pro) in the ADAM12 prodomain results in the complete retention of the enzyme in ER (Cao et al., 2002). Sucrose gradient fractionation of brain lysates revealed that the mature forms of ADAM10 and APP are enriched in lipid raft fractions, where ectodomain shedding of ADAM10 itself and α-secretase cleavage of APP mainly occur (Figure 7E). However, neither the prodomain LOAD mutations nor DN mutations altered the cellular trafficking of ADAM10 and APP to ER and lipid rafts. Surface biotinylation

of primary cortical neurons derived from ADAM10 transgenic mouse embryos also indicated that the Q170H prodomain mutation did not change the trafficking of the enzyme to the plasma Obeticholic Acid in vitro membrane (Figures 7F and 7G), a major location responsible for APP cleavage by α-secretase. We also examined whether the prodomain mutations affect ADAM10 trafficking to the synapse, in which the activity of the metalloprotease is regulated by synapse-associated protein-97 (SAP-97) (Marcello et al., 2007). As shown in Figure 7H, compared to the whole-brain homogenates, the levels of both APP and APP-CTFα were elevated in synaptosomal fraction and LOAD mutations decreased APP-CTFα levels. However, ADAM10 levels at the synapse were not changed by the prodomain mutations. Together, these results suggest that the attenuated α-secretase cleavage

of APP by the LOAD mutations is not caused by altered ADAM10 trafficking. We next tested whether the LOAD ADAM10 mutations affect the prodomain chaperone function. Previous studies have shown that addition of a prodomain in Levetiracetam trans to a prodomain-deleted enzyme enables the completion of protein folding and restores the enzyme activity for many types of proteases, including ADAM10 ( Anders et al., 2001 and Cao et al., 2000). Thus, we asked whether the ADAM10 prodomain in trans affects the activity of prodomain-deleted ADAM10 (ADAM10Δpro). We also tested whether the two prodomain mutations affect the chaperone activity of ADAM10 prodomain as compared to WT. To this end, neuroblastoma H4 cells stably overexpressing APP were transfected with either ADAM10Δpro alone or in combination with ADAM10 prodomain constructs expressing WT, Q170H, or R181G forms of ADAM10.