AMPA receptor contribution to methylmercury-mediated alteration of intracellular Ca2+ concentration in human induced pluripotent stem cell motor neurons

Abstract

α motor neurons (MNs) are a target of the environmental neurotoxicant methylmercury (MeHg), accumulating MeHg and subsequently degenerating. In mouse spinal cord MN cultures, MeHg increased intracellular Ca²⁺ [Ca²⁺]_i; the AMPA receptor (AMPAR) antagonist CNQX delayed the increase in [Ca²⁺]_i, implicating the role of AMPARs in this response. Here we used human induced pluripotent stem cell-derived MNs (hiPSC-MNs), to characterize the role of MN AMPARs in MeHg neurotoxicity. Acute exposure to MeHg (0.1, 0.2, 0.5, 1 and 1.5 μM), fura-2 microfluorimetry, and a standard cytotoxicity assay, were used to examine MN regulation of [Ca²⁺]_i, and cytotoxicity, respectively. Contribution of Ca²⁺-permeable and impermeable AMPARs was compared using either CNQX, or the Ca²⁺-permeable AMPAR antagonist N-acetyl spermine (NAS). MeHg-induced cytotoxicity was evaluated following a 24 h delay subsequent to 1 h exposure of hiPSC-MNs. MeHg caused a characteristic biphasic increase in [Ca²⁺]_i, the onset of which was concentration-dependent; higher MeHg concentrations hastened onset of both phases. CNQX significantly delayed MeHg's effect on onset time of both phases. In contrast, NAS significantly delayed only the 2nd phase increase in fura-2 fluorescence. Exposure to MeHg for 1 h followed by a 24 h recovery period caused a concentration-dependent incidence of cell death. These results demonstrate for the first time that hiPSC-derived MNs are highly sensitive to effects of MeHg on [Ca²⁺]_i, and cytotoxicity, and that both Ca²⁺-permeable and impermeable AMPARs contribute the elevations in [Ca²⁺]_i.

Keywords: AMPA receptor; Glutamate; Intracellular calcium; Ion channel; Methylmercury; Motor neurons.