Fig. 1.

Effects of 5-HIAA on NEP expression and activity in SH-SY5Y neuronal cells and mouse brain. a, b, c: Experiments in SH-SY5Y neuroblastoma cells. a Kinetic of NEP activity as a function of incubation time (0 to 180 min) in cells treated by 100 μM 5-HIAA. NEP activity was determined with a fluorescent SensoLyte 520 kit (AnaSpec Inc., Fremont, CA, US). NEP activity culminates at 90–120 min after addition of the compound, with quantitative values corresponding to 20 +/− 1 and 19 +/ 2 10³ DO/mg of total proteins, respectively. These maximal values decrease to basal level after 3 h. b q-PCR assessment of NEP mRNA increase as a function of incubation time (0 to 90 min) in SH-SY5Y cells treated by 100 μM 5-HIAA. The increase of NEP mRNA level already detectable at 30 min, reached a value 30 times higher at 90 min which represents the time point corresponding to NEP maximal activity. c ELISA test determination of the dose-response effect of 24 h 5-HIAA treatment on NEP activity. Evaluation of the effects of increasing doses of 5-HIAA showed an EC₅₀ of 8 ± 3 μM, a concentration within the range of 5-HIAA concentration found in brain tissue. The statistical analysis of triplicate samples was done by an ANOVA completed with a Bonferroni’s Multiple Comparison Test. ** p < 0.005, *** p < 0.0001. d Immunocytochemistry with anti-NEP antibody of SH-SY5Y cells treated with 100 μM 5-HIAA for 24 h versus non-treated controls. Scale bars: 20 μm. e Fluorescence-activated cell sorting (FACS) by flow cytometry on SHSY-5Y cells treated with 5-HIAA (100 μM) for 24 h versus controls. 46% of cells were neprilysin-positive in control cells while 89% were positively labeled in treated cells. Black curve: no secondary antibody; Red curve: cells treated with NEP secondary antibody. f, g: Experiments on mouse brain in vivo. f Effects of 5-HTP (48 mg/kg, I.P.) or 5-HIAA (24 μL of a 30 mM solution, intra-nasal) on NEP activity in 14 months APPSWE mice brain (n = 5 for each treatment). Drugs were administered each day during five consecutive days. Both 5-HTP and 5-HIAA treatments increase the level of NEP activity in brain (142 +/− 7 and 192 +/− 18 Relative Fluorescence Units (RFU)/min, respectively). However, 5-HIAA treatment was the most effective. g Kinetics of NEP enzymatic activity in 3 months Swiss Albinos mice (n = 5 at each time point) treated by an intra-nasal administration of 24 μL of a 30 mM solution of 5-HIAA at time zero. The brain NEP activity was determined every 30 min until 120 min. A rapid increase of NEP activity was detected at 90 min with a maximum value of 2040 +/− 50 RFU/min. The statistical analysis was done by an ANOVA completed with a Bonferroni’s Multiple Comparison Test. * p < 0.05; ** p < 0.005; *** p < 0.0001 by reference to NT or time zero