The DLK- and AP-1-dependent signaling pathway controlling App gene transcription and Aβ-synthesis is conserved in mouse neuronsAll experiments were carried out in dissociated mixed mouse neuron/glia cultures in which endogenous glia factors maximally stimulate ApoE-dependent signaling pathways.(A) Exogenous ApoE3 has no effect on APP mRNA levels in neuron/glia cultures from mouse cortex, but inhibition of DLK by MBIP overexpression decreases APP mRNA levels, whereas DLK or MKK7 overexpression increase APP mRNA levels. Neuron/glia cultures were transduced with lentiviruses at DIV4, treated with ApoE3 (10 µg/ml) at DIV10, and analyzed by RT-PCR at DIV12.(B) DLK knockdown decreases APP and DLK protein levels in neuron/glia cultures from mouse hippocampus, and additionally suppresses steady-state phosphorylation of ERK1/2 and MKK7, whereas rescue overexpression of either DLK or MKK7 increases APP protein levels and MKK7 and ERK1/2 phosphorylation.(C & D) DLK knockdown decreases APP mRNA levels (C) and Aβ secretion (D) in neuron/glia cultures from mouse hippocampus, whereas rescue overexpression of either DLK or MKK7 increases APP mRNA levels (C) and Aβ40 and Aβ42 secretion (D).(E) Alignment of the human APP- and murine App-promoter sequences containing the AP-1 binding site demonstrates a high degree of conservation.(F & G) CRISPRi-mediated inhibition of AP-1 binding to the App-promoter in neuron/glia cultures from mouse hippocampus suppresses APP mRNA (F) and protein levels (G).(H) CRISPRi-mediated inhibition of AP-1 binding to the App-promoter decreases Aβ40 and Aβ42 secretion in neuron/glia cultures from mouse hippocampus.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 comparing to control with Tukey’s post-hoc multiple comparisons [(A) to (D)] and Student’s t test [(F) to (H)]. For additional data, see Fig. S7.
