ApoE-mediated activation of cFos-containing transcription factor AP-1 stimulates APP-gene transcription(A) CRISPRi strategy to identify APP promoter sequences required for ApoE-stimulation of APP-transcription using guide RNAs (sg1 to sg6) covering the proximal human APP-promoter (note that sg2 targets conserved AP-1 binding site).(B) CRISPRi of AP-1 binding sequence in human APP-promoter blocks ApoE3-induced increase in APP mRNA levels. Human neurons on MEFs were infected at D4 with lentiviruses co-expressing BFP-tagged dCas9, various sgRNAs, and mCherry. Neurons were treated at D10 with ApoE3 (10 µg/ml), and APP mRNA levels were measured at D12.(C) CRISPRi of AP-1 binding sequence in human APP-promoter blocks ApoE3-induced increase in APP protein but not in ERK1/2-phosphorylation. Experiments were performed as for B, except that neurons were analyzed by quantitative immunoblotting (left, representative blots; right, summary graph).(D) CRISPRi of AP-1 binding sequence in human APP-promoter suppresses ApoE-induced, but not basal Aβ42 secretion from human neurons on MEFs. Experiments were performed as described for (B).(E) ApoE activates cFos phosphorylation in human neurons co-cultured with MEFs with an ApoE4>ApoE3>ApoE2 rank potency order; cFos phosphorylation is blocked by the ApoE-receptor antagonist RAP (top, representative immunoblots; bottom, summary graphs).(F) AP-1 binding site in human APP-promoter mediates ApoE stimulation of APP-gene transcription. Human neurons cultured on MEFs were infected at D4 with lentiviruses containing APP-promoter-driven firefly luciferase and constitutively expressed renilla luciferase (internal control), treated with ApoE (10 µg/ml) at D10, and analyzed at D12 (top, schematic of promoter reporter construct; bottom, summary graph of luciferase expression normalized to the renilla luciferase control).(G & H) Dominant-negative cFos (DN-cFos) suppresses ApoE-induction of APP mRNA levels (G) and APP protein (H).Data are means ± SEM (n≥3 independent experiments for all bar graphs); statistical significance (*, p<0.05, **, p<0.01; ***, p<0.001; n.s., not significant) was evaluated with one-way ANOVA with Tukey’s post-hoc test or Student’s t-test. In (B) and (C), the difference between –ApoE3 and +ApoE3 is significant for control, sg1 and 3–6 groups (p<0.001 as indicated), but not for sg2 (n.s.). For additional data, see Fig. S6.
