Methylmercury impairs neurotrophin signaling and disrupts neuronal differentiation

Methylmercury (CH₃Hg) is a potent developmental neurotoxicant but the underlying mechanism of action is unclear. Because neurotrophin signaling is important for differentiation and survival in the developing nervous system, we examined the hypothesis that CH₃Hg could disrupt differentiation via actions on the neurotrophin signal transduction cascade. Using pheochromocytoma (PC12) cells, we examined the effects of CH₃Hg on differentiation, survival and neurotrophin signaling. In primed PC12 cells following 24 hr exposure to 50 ng/ml Nerve Growth Factor (NGF) and various concentrations of CH₃Hg, there was a concentration-dependent inhibition of neurite outgrowth with an EC₅₀ of approximately 0.03 μM. At higher concentrations (1–10 μM), CH₃Hg-induced cell death through an apoptotic pathway. TrkA activation by autophosphorylation was determined by immunoblotting with a phospho-specific antibody. Concurrent exposure to CH₃Hg and NGF for 2.5 min resulted in a concentration-dependent decrease in TrkA autophosphorylation, which was significant at 100 nM CH₃Hg. NGF-induced phosphorylation of Extracellular Regulated Kinase (ERK) 1/2 in PC12 cells was measured by immunoblotting with a phospho-specific antibody. Concurrent exposure to CH₃Hg and NGF for 2.5 min resulted in a concentration-dependent inhibition of ERK 1/2 activation, which was significant at 100 nM CH ₃Hg. CH₃Hg did not directly inhibit ERK or the upstream kinase, MAP and ERK Kinase (MEK). In PC12 cell homogenates, CH₃Hg produced a concentration-dependent inhibition of Protein Kinase C (PKC) activity that was significant at 0.3–10 μM and inhibited PKCδ, ϵ, and ζ activity in a concentration-dependent manner at higher concentrations (3–10 μM), while a significant increase in PKCα activity was observed at lower concentrations (0.03 μM). However, CH₃Hg did not affect NGF-induced activation or translocation of PKC in intact cells. To determine whether the observed CH₃Hg-induced inhibition of TrkA and ERK 1/2 activation was sufficient to alter cell differentiation, NGF-stimulated neurite outgrowth was examined after exposure to selective Trk inhibitor K252a (30 nM) or selective MEK inhibitor U0126 (300 nM). A 24 h exposure to CH ₃Hg, K252a or U0126 reduced neurite outgrowth to a similar degree. Our results suggest that CH₃Hg may inhibit differentiation of PC12 cells by interfering with NGF-stimulated TrkA autophosphorylation and signaling via the MAPK cascade.