Skip to main content
. Author manuscript; available in PMC: 2018 Jan 26.
Published in final edited form as: Cell. 2017 Jan 19;168(3):427–441.e21. doi: 10.1016/j.cell.2016.12.044

Figure 4. ApoE increases APP expression 3-4-fold with an ApoE4>ApoE3>ApoE2 potency rank order by activating the DLK→MKK7→ERK1/2 MAP-kinase pathway.

Figure 4

(A) Human neurons synthesize ~5-fold less APP when cultured on MEFs instead of glia; addition of ApoE2, ApoE3, or ApoE4 (each 10 µg/ml applied from D10-12) stimulates APP synthesis in human neurons on MEFs with a ApoE4>ApoE3>ApoE2 potency rank order, which is blocked by the ApoE-receptor antagonist RAP (left, representative immunoblots; right, summary graphs; see also Fig. S5).

(B) ApoE2, ApoE3, and ApoE4 increase APP mRNA levels 3–4 fold with a ApoE4>ApoE3>ApoE2 potency rank order in human neurons cultured on matrigel only.

(C) ApoE2, ApoE3, and ApoE4 increase only APP, but not APLP1 or APLP2 mRNA levels in human neurons on MEFs with an ApoE4>ApoE3>ApoE2 potency rank order; ApoE-induced APP mRNA increase is inhibited by RAP and the MAP-kinase inhibitor U0126, but not by the PI3K inhibitor Wortmannin.

(D) Recombinant cholesterol-free ApoE2, ApoE3, and ApoE4 produced in bacteria stimulate APP mRNA levels in human neurons on MEFs similar to recombinant ApoE2, ApoE3, and ApoE4 produced in HEK293 cells.

(E) Inhibition of DLK by shRNAs or MBIP blocks ApoE3-induced increases in APP protein levels, while DLK and MKK7 overexpression during rescue experiments constitutively increases APP protein levels independent of ApoE3. Note that DLK protein levels were not affected by MKK7 manipulations (left, representative blots; right, summary graphs).

(F) Knockdown of the JNK scaffold JIP3 has no effect on ApoE3-induced activation of the DLK→MKK7→ERK1/2 signal transduction cascade, but blocks induction of the JNK MAP-kinase cascade during the stress response to MG132. Human neurons on MEFs were infected with lentiviruses expressing a control shRNA or a JIP3 shRNA at D4, treated with control medium, ApoE3 (10 µg/ml), or MG132 (0.1 g/l) for 2 hours at D10, and analyzed by immunoblotting (left, representative blots; right, summary graphs).

Data are means ± SEM (n≥3 independent experiments for all bar graphs); 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 comparison (A–D, F) or comparisons to controls (E). For further data and controls, see Fig. S4, S5.