Effects of thimerosal on NGF signal transduction and cell death in neuroblastoma cells.

Signaling through neurotrophic receptors is necessary for differentiation and survival of the developing nervous system. The present study examined the effects of the organic mercury compound thimerosal on nerve growth factor signal transduction and cell death in a human neuroblastoma cell line (SH-SY5Y cells). Following exposure to 100 ng/ml NGF and increasing concentrations of thimerosal (1 nM-10 microM), we measured the activation of TrkA, MAPK, and PKC-delta. In controls, the activation of TrkA MAPK and PKC-delta peaked after 5 min of exposure to NGF and then decreased but was still detectable at 60 min. Concurrent exposure to increasing concentrations of thimerosal and NGF for 5 min resulted in a concentration-dependent decrease in TrkA and MAPK phosphorylation, which was evident at 50 nM for TrkA and 100 nM for MAPK. Cell viability was assessed by the LDH assay. Following 24-h exposure to increasing concentrations of thimerosal, the EC50 for cell death in the presence or absence of NGF was 596 nM and 38.7 nM, respectively. Following 48-h exposure to increasing concentrations of thimerosal, the EC50 for cell death in the presence and absence of NGF was 105 nM and 4.35 nM, respectively. This suggests that NGF provides protection against thimerosal cytotoxicity. To determine if apoptotic versus necrotic cell death was occurring, oligonucleosomal fragmented DNA was quantified by ELISA. Control levels of fragmented DNA were similar in both the presence and absence of NGF. With and without NGF, thimerosal caused elevated levels of fragmented DNA appearing at 0.01 microM (apoptosis) to decrease at concentrations >1 microM (necrosis). These data demonstrate that thimerosal could alter NGF-induced signaling in neurotrophin-treated cells at concentrations lower than those responsible for cell death.

Methylmercury inhibits TrkA signaling through the ERK1/2 cascade after NGF stimulation of PC12 cells.

Using PC12 cells as a model of neuronal differentiation, we have shown that acute exposure to methylmercury (CH3Hg) inhibits nerve growth factor (NGF)-induced activation of TrkA. In the present study, we examined the effects of CH3Hg on pathways activated by NGF. NGF-induced phosphorylation of ERK1/2 in PC12 cells was time-dependent. Concurrent exposure to CH3Hg and NGF for 2.5 min resulted in a concentration-dependent inhibition of ERK1/2 phosphorylation (EC50 = 0.018 microM). However, NGF-stimulated ERK1/2 phosphorylation was not altered after 5 min of exposure to CH3Hg. In vitro studies revealed that CH3Hg did not directly inhibit the ERK kinase MEK. As reported in other neuronal tissue, CH3Hg can inhibit PKC activity in vitro. Incubation of PC12 cell lysates with CH3Hg produced a concentration-dependent inhibition of PKC activity that was significant at 0.3-10 microM. Further studies using recombinant enzymes examined the effect of CH3Hg on PKC isoforms expressed in PC12 cells. CH3Hg inhibited PKCdelta, and zeta activity in a concentration-dependent manner at higher concentrations (3-10 microM), while a significant increase in PKCalpha activity was observed at lower concentrations (0.1 microM). However, CH3Hg had no affect on NGF-induced PKC activity in intact cells. These results show that CH3Hg inhibition of NGF-stimulated TrkA activation in PC12 cells decreases downstream signaling through the Raf/MEK/ERK cascade. In intact cells PKC does not appear to be a primary target for CH3Hg.

Methylmercury decreases NGF-induced TrkA autophosphorylation and neurite outgrowth in PC12 cells.

Neurotrophin signaling through Trk receptors is important for differentiation and survival in the developing nervous system. The present study examined the effects of CH(3)Hg on (125)I-nerve growth factor (NGF) binding to the TrkA receptor, NGF-induced activation of the TrkA receptor, and neurite outgrowth in an in vitro model of differentiation using PC12 cells. Whole-cell binding assays using (125)I-NGF revealed a single binding site with a K(d) of approximately 1 nM. Methylmercury (CH(3)Hg) at 30 nM (EC(50) for neurite outgrowth inhibition) did not affect NGF binding to TrkA. TrkA autophosphorylation was measured by immunoblotting with a phospho-specific antibody. TrkA autophosphorylation peaked between 2.5 and 5 min of exposure and then decreased but was still detectable at 60 min. Concurrent exposure to CH(3)Hg and NGF for 2.5 min resulted in a concentration-dependent decrease in TrkA autophosphorylation, which was significant at 100 nM CH(3)Hg. To determine whether the observed inhibition of TrkA was sufficient to alter cell differentiation, NGF-stimulated neurite outgrowth was examined in PC12 cells after exposure to 30 nM CH(3)Hg, a concentration that inhibited TrkA autophosphorylation by approximately 50%. For comparison, a separate group of PC12 cells were exposed to a concentration of the selective Trk inhibitor K252a (30 nM), which had been shown to produce significant inhibition of TrkA autophosphorylation. Twenty-four hour exposure to either CH(3)Hg or K252a reduced neurite outgrowth to a similar degree. Our results suggest that CH(3)Hg may inhibit differentiation of PC12 cells by interfering with NGF-stimulated TrkA autophosphorylation.