Figure 5.

The phosphorylation of SAP97 at T629 is reduced in AD patients' hippocampi and affects ADAM10 localization/activity. (a) IP carried out from HCs and AD patients post-mortem hippocampi homogenates (HOMO) with an Ab raised against phosphorylated T629 (anti-SAP97-T629P). WB analysis was performed with SAP97 Ab. SAP97 T629 phosphorylation is reduced in AD patients compared with HCs. Leftmost lanes, WB performed on HCs and AD hippocampi HOMO showed no alterations of SAP97 levels. (b) Quantification of IP experiments in (a) (n=12, *P=0.012 AD versus HCs, paired t-test). (c) Representative WB of sAPPα and total sAPP released by human embryonic kidney 293 (HEK293) cells stably expressing APP and transfected with either YFP-SAP97wt or phosphomimetic mutants. SAP97wt expression stimulates ADAM10 activity, and the mutant mimicking T629 phosphorylation further increases this effect. (d) Quantitative analysis of experiments in (c). The levels of sAPPα were normalized on total sAPP and data are expressed as the percentage of mock cells (n=4, *P<0.05 versus mock; ^#P<0.05 versus YFP-SAP97-T629D, one-way analysis of variance (ANOVA), Bonferroni's post hoc test). (e) COS-7 cells were transfected with ADAM10 full-length (fl) alone or co-transfected with either YFP-SAP97wt or phosphomimetic mutants and stained for ADAM10 surface/total expression. ADAM10fl alone was faintly localized at the surface despite intense intracellular labeling. In contrast, the co-transfection of ADAM10fl and YFP-SAP97wt or the mutants increased ADAM10fl surface expression. Notably, the YFP-SAP97-T629D mutant had the strongest effect on ADAM10fl surface expression. Scale bar, 10 μm. (f) Quantification of experiments in (e) (*P<0.05 versus ADAM10fl; ^#P<0.05 versus YFP-SAP97-T629D; 16 cells per condition from two independent experiments; one-way ANOVA, Bonferroni's post hoc test)