Human neurons express high levels of the MAP-kinase kinase kinase DLK (Fig. S2C, S2D), which is an attractive candidate for neuronal signaling because it has been implicated in axonal regeneration, synaptogenesis, and neurodegeneration (reviewed in Tedeschi and Bradke, 2013; Lu et al., 2014). Indeed, we found that ApoE induced a 2- to 4-fold increase in DLK protein levels in human neurons co-cultured with MEFs; this induction again was blocked by RAP, which had no effect on baseline DLK levels (Fig. 2C). Again, the three human ApoE isoforms increased DLK levels with the same differential potency as for the stimulation of Aβ synthesis and ERK1/2 phosphorylation (ApoE4>ApoE3>ApoE2; Fig. 2C). Neurons expressed high levels of DLK mRNA, but newly synthesized DLK protein was rapidly degraded (Fig. 2E). Proteasome inhibition with MG132 dramatically increased DLK protein levels under control conditions, and occluded subsequent ApoE3-induced increases of DLK levels (Fig. 2D). ApoE3 had no effect on phosphorylation of JNK1/2/3, the most abundant MAP-kinase in neurons, although JNK1/2/3 phosphorylation was stimulated by MG132, which induces a cellular stress response that activates JNKs (Fig. 2D).
