Figure 5.

Proinflammatory cytokine-mediated influence of hμG on hRGC neuritogenesis involves the mTOR pathway

Western blot analysis carried out on the cell lysates of hRGCs in four different culture conditions to examine the specificity of TNF-α and IL-1β influence and recruitment of the AKT/mTOR pathway (A). Levels of immunoreactivities corresponding to pAKT, pmTOR, and pS6 increased significantly in the presence of hμG CM versus controls, which is abrogated in antibody-neutralized hμG CM (B–D), demonstrating the engagement of the AKT/mTOR pathway by TNF-α and IL-1β in hμG CM. Inclusion of rapamycin in hμG:hRGC co-culture decreased the levels of pAKT, pmTOR, and pS6 more than twice the levels observed in the presence of hμG CM/hμG CM with neutralizing antibody, corroborating the engagement of mTORC1 (A–D). Both the length and the complexities of SMI32⁺ TAU1⁺ hRGC axons decreased significantly in the presence of rapamycin compared to those cultured in the presence of hμG CM and antibody-neutralized hμG CM demonstrating that proinflammatory cytokine-mediated influence of hμG on neuritogenesis involves the AKT/mTOR pathway (E-J). That the AKT/mTOR-mediated effects on neuritogenesis may engage intrinsic mechanism for neurite growth was demonstrated by a decrease in the levels of GAP43, SOX11, and KLF6 transcripts in the presence of rapamycin versus all other groups (K-M). Experiments were carried out in triplicates per group from 3 independent biological replicates. Values expressed as mean ± SEM. Scale bar: 50 μm.