The ApoE-induced increase in APP mRNA levels was blocked by RAP and by U0126 but not by Wortmannin, suggesting that it is dependent on ApoE-binding to surface receptors and on ApoE-induced activation of MAP-kinases (Fig. 4A, 4C). Previous studies suggested that ApoE-containing lipoproteins activate neurons by providing cholesterol (Mauch et al., 2001). Since ApoE protein produced in transfected HEK293 cells is secreted as a lipidated particle containing cholesterol, we tested cholesterol-free ApoE produced in E. coli, but observed the same 3- to 5-fold enhancement in APP mRNA levels with the same ApoE4>ApoE3>ApoE2 potency rank order as with HEK293-cell derived ApoE (Fig. 4D, S1C). Thus, ApoE acts by a cholesterol-independent, direct receptor action, as also suggested by its sensitivity to RAP. Moreover, we observed that inhibition of DLK by shRNAs or MBIP overexpression and inhibition of MKK7 by CRISPR both blocked the ApoE-induced increase in APP protein (Fig. 4E, S4D–S4F). DLK or MKK7 overexpression, conversely, constitutively increased APP mRNA and protein levels (Fig. 4E). Thus, activation of surface ApoE-receptors activates a non-canonical DLK→MKK7→ERK signaling pathway that increases APP mRNA and APP protein levels.
