Figure 8.
LM11A compounds induce survival signaling similar to, but distinct from, neurotrophins. A, Survival of hippocampal neurons in cultures treated with signaling pathway inhibitors and BDNF (0.95 nm), NGF (0.95 nm), or LM11A compounds (5 nm), showing substantial inhibition by NFκB and PI3K pathway inhibitors, small effects of ERK inhibition on BDNF and NGF activity, and no effect of ERK inhibition on LM11A compound activity. SN50, An NFκB translocation inhibitor; LY, LY294002, a PI3K inhibitor; PD, PD98059, an ERK inhibitor. n = 18 for each bar, showing mean + SE; NS, not significant; *p < 0.05, **p < 0.001 for comparison with control (no inhibitor) in each group, Student’s t test. B–F, Western blot analysis of extracts of hippocampal cultures. Top, Representative bands corresponding to phosphorylated signaling factors (open arrowheads) and the corresponding total factor (filled arrowheads); graphs, quantitation of the ratio of phospho- to total factor, indicating degree of activation. B, NFκB-p65 activation analysis, showing similar activation kinetics for all biologically active treatments. Cells were treated with culture media (C), BDNF (B) at 1.9 nm, NGF (N) at 1.9 nm, and LM11A compounds at 20 nm. The bars indicate mean + SEM. Black bars, Sampling at 10 min; gray bars, 30 min. n = 6 independent blots for each determination. *p < 0.05, **p < 0.005, ***p < 0.001 for comparison with culture medium only by Student’s t test. C, LM11A-31 dose–response of NFκB pathway activation. Bars indicate mean + SE; n > 6 for each condition. p values as indicated, Student’s t test. Activation is detected between 0 and 0.5 nm and reaches a plateau at 5 nm. D, AKT activation analysis as in B, showing a small lag in activation by the active compounds relative to NGF. E, LM11A-31 dose–response of AKT pathway activation as in C. Activation is detected between 0.5 and 1 nm and reaches a plateau level at 5 nm. F, AKT activation by growth factors and compounds in p75NTR−/− cells. n > 9 for each condition. There are no significant differences between culture medium alone and NGF or the LM11A compounds.