In our experiments, the culture of human neurons maintained independent of glia or serum allowed unprecedented control over exogenous signals acting on these neurons. Moreover, application of CRISPRi to map transcription-factor-binding sequences in a promoter in cultured cells and in vivo might prove generally useful for mapping promoter elements. Conceptually, the finding that ApoE activates a non-canonical MAP-kinase pathway involving ERK1/2 phosphorylation by MKK7 but not JNKs, and that ApoE acts here in a receptor-dependent, cholesterol-independent manner were surprising. The upstream mechanism involved – stabilizing DLK by preventing its rapid proteasomal degradation – was also unexpected, as was the downstream effector pathway whereby cFos-phosphorylation stimulates APP transcription via a specific AP-1 binding sequence that is evolutionarily conserved and that functions in vivo. Remarkably, ApoE upregulated only APP, but not its close homologs APLP1 and APLP2. Thus, our studies describe an unanticipated signal transduction pathway that links ApoE-signaling to Aβ-synthesis in human neurons.
