ApoE stimulates a non-canonical DLK→MKK7→ERK1/2 MAP-kinase signalling cascade in human neurons(A) ApoE (10 µg/ml, applied at D10 for 2 h) activates ERK1/2 phosphorylation in human neurons cultured on MEFs with an ApoE4>ApoE3>ApoE2 potency rank order (left, representative immunoblots; right, summary graphs).(B) ApoE3-induced ERK1/2-phosphorylation is abolished by the ApoE-receptor blocker RAP and by inhibitors of MEKs (PD9* [PD98059] and U0126; both 50 µM), but not by inhibitors of JNK (SP6* [SP600125]; 25 µM), PI3K kinase (Wortmannin; 0.1 µM), or Src kinase (PP2; 10 µM). Drugs were applied 30 min before a 2 h ApoE3 (10 µg/ml) incubation at D10. For additional data, see Fig. S2.(C) ApoE2, ApoE3, and ApoE4 cause a rapid, 2–3 fold increase in DLK in human neurons cultured on MEFs with an ApoE4>ApoE3>ApoE2 potency rank order; recombinant RAP that blocks ApoE-receptor binding also blocks ApoE-induced increases in DLK (left, representative immunoblots; right, summary graphs from neurons at D10 treated for 2 h with 10 µg/ml ApoE).(D) Proteasome inhibitor MG132 (10 µM, applied for 2 h at D10) increases DLK in human neurons cultured on MEFs similar to ApoE3 (10 µg/ml), thereby occluding the effect of ApoE3 (left); in addition, MG132 stimulates JNK phosphorylation (right). The transcription inhibitor actinomycin D (1 µg/ml) has no effect on the ApoE-induction of DLK (left) or JNK phosphorylation (right).(E) ApoE3 significantly slows the rapid turnover of DLK protein (measured by immunoblotting after addition of the protein synthesis inhibitor cycloheximide (0.1 g/l); left, representative blot; center, summary plots of the fraction of DLK remaining as a function of time after cycloheximide addition; right, summary graphs of the DLK decay rates and calculated half-lives as a function of ApoE3).(F) ApoE3 has no effect on ERK1/2 protein levels that are stable, but ApoE3-stimulated ERK1/2 phosphorylation decays in parallel with DLK protein levels after protein synthesis inhibition (left, summary plots of the fraction of ERK1/2 remaining as a function of time after cycloheximide addition; right, summary plot of the phospho-ERK/total ERK ratio).(G) ApoE3 strongly stimulates MKK7 and ERK1/2 phosphorylation but not JNK phosphorylation in human neurons cultured on MEFs (at D10); DLK knockdown with an shRNA or DLK inhibition by MBIP overexpression block ApoE3-induced MKK7 and ERK1/2 phosphorylation, whereas DLK overexpression constitutively activates MKK7 and ERK1/2 phosphorylation (left, representative immunoblots; right, summary graphs).Data are means ± SEM (n≥3 independent experiments); statistical significance (*, p<0.05, **, p<0.01; ***, p<0.001) was evaluated with one-way ANOVA and Tukey’s post-hoc test in pairwise comparisons in A and C and comparisons to control in B and D, and with two-way ANOVA in E and F.
