The fact that the proteasome inhibitor MG132 mimics and occludes the effect of ApoE on DLK levels suggests that ApoE increases DLK levels by decreasing rapid proteasomal degradation of DLK. To test this hypothesis, we blocked new protein synthesis in human neurons with cycloheximide in the presence and absence of ApoE3, and measured the decay rate of previously synthesized DLK and ERK1/2 proteins, as well as the degree of ERK1/2 phosphorylation (Fig. 2E, 2F). We found that DLK was indeed rapidly degraded in the absence of ApoE3, whereas ERK1/2 were relatively stable. ApoE3 significantly decreased the DLK decay rate, but had no effect on ERK1/2 protein levels (Fig. 2E, 2F). As above, ERK1/2 phosphorylation was strongly stimulated by ApoE3, and decayed in parallel with DLK levels after cycloheximide addition (Fig. 2F, S2E). Thus, ApoE activates DLK by decreasing its rapid proteasomal degradation.
