Fig. 4

5-HIAA induces NEP expression via the MAP-kinase/ERK ½ pathway. a Down-regulatory effect of 5-HIAA on ERK ½, MEK ½ and GSK-3 expression in SH-SY5Y cells after 30 min treatment. The ERK pathway was down-regulated by 5-HIAA (mean decrease of ERK1–17% and ERK2–21%). GSK-3 α/β, a strategic factor for tau phosphorylation, was also significantly affected by the treatment (mean decrease of GSK-3α/β and GSK-3β − 15%), together with the transcription factor CREB (mean decrease − 40%). The statistical analysis of triplicate samples was done with a Student t-test. * p < 0.05, ** p < 0.005 by reference to non-treated cells (NT). b Effects of separated or combined treatments with the ERK ½ inhibitor SCH772984 and/or 5-HIAA on NEP expression in SH-SY5Y cells after 90 min treatment. NEP protein expression was stimulated by SCH772984 (26 +/− 4%) or 100 μM 5-HIAA (49 +/− 2%) tested separately. The combined treatment containing 100 μM 5-HIAA and SCH772984 showed a higher increase percentage of NEP expression (63 +/− 2%) compared to the separate treatments. The statistical analysis of triplicate samples was done by an ANOVA completed with a Bonferroni’s Multiple Comparison Test. ^## p < 0.005, ^### or *** p < 0.0001. * by reference to non-treated cells (NT), or ^# by reference to treatments with 5-HIAA. c Inhibitory action of 5-HIAA on AICD expression in SH-SY5Y cells. NH₄Cl as positive control. The statistical analysis (n = 5 in each group) was performed by an ANOVA completed with a Bonferroni’s Multiple Comparison Test. *** p < 0.0001 by reference to non-treated cells (NT). d Dose-response effect and EC₅₀ of SCH772984 and Trametinib on NEP expression in SH-SY5Y cells. Both MEK inhibitor (GSK 1120212, Trametinib) and ERK kinase inhibitor (SCH772984) induce NEP with an EC₅₀ of 14 +/− 4 and 27 +/− 3 nM, respectively. e Graphical summary of potential mechanisms involved in 5-HIAA-induced NEP expression. The MAP-kinase pathway is involved in NEP expression in SH-SY5Y cells: inhibition of MEK/ERK ½ phosphorylation increased NEP protein levels. Phosphorylation of GSK-3 is also down-regulated by 5-HIAA treatment. These effects may explain 5-HIAA-induced increase in Aβ peptide clearance