Figure 1

(a) Effects of JNK inhibition on APP processing in cortical neurons. Cortical neurons were pre-treated with 2, 4 and 6 μM of D-JNKI1 for 24 h and cell lysates were immunoblotted for APP (22C11 antibody), βAPPs, αAPPs and P-APP (Thr⁶⁶⁸). Loading control, tubulin. There were dose-dependent reductions of APP (m=mature APP and im=immature APP), βAPPs, and αAPPs production and APP phosphorylation. Proteins in the culture media were precipitated and immunoblotted for APPs and the peptide reduced the APPs level. (b) Western blot densitometry clearly showed that the peptide reduced APPs, βAPPs, αAPPs and P-APP levels. Quantifications were from eight independent experiments (±S.E.M.); ^*P<0.05 versus control. (c) Quantitative determination of beta-amyloid fragments (1–40 and 1–42) in culture media by ELISA assay. The peptide had the same effect on Aβ 1–40 and Aβ 1–42 (10% decrease with 2 mM D-JNKI1, 20% with 4 mM and 30% with 6 mM). Data are mean of eight independent experiments (±S.E.M.), ^*P⩽0.05 versus control. (d) P-APP and P-c-jun immunofluorescence. Left-hand panels show control neurons, right-hand panel neurons treated with 4 μM D-JNKI1 for 24 h, after which the neurons were stained with P-APP (Thr⁶⁶⁸) antibody, red (upper panels) and P-c-Jun antibody, red (lower panels), and nuclei were counterstained with syto13 reagent, green. In the control condition, P-APP labelling was diffusely distributed in soma, dendrites and nuclei of cortical neurons. D-JNKI1 strongly reduced the labelling, limiting it to the perinuclear and nuclear regions. P-c-jun staining was strongly reduced by D-JNKI1 pre-treatment. Scale bar, 50 μm. (e) APP degradation. D-JNKI1 treatment (6 μM) induced APP degradation (65%), whereas co-treatment with MG132 (5 μM) prevented it (reducing APP degradation to 20%). The calpain inhibitor calpastatin had no such effect (2 μM). Quantification was carried out on six independent experiments (±S.E.M.); ^*P<0.05 versus control. In red, statistical comparison between D-JNKI1 and (1) the co-treatment D-JNKI1/MG132 and (2) D-JNKI1/calpastatin; red asterisk=significant difference determined by Tukey's test. (f) APP degradation. Co-treatment with Z-vad (10 μM) and MG132 had minor effect on APP degradation (35%). Red lines indicate the comparison between D-JNKI1/MG132 and z-vad/MG132 by Tukey's test: there is no significant difference. Quantification was carried out on three independent experiments (±S.E.M.); ^*P<0.05 versus control