AMPA receptor contribution to methylmercury-mediated alteration of intracellular Ca 2+ concentration in human induced pluripotent stem cell motor neurons

Neurotoxicology. 2020 Dec;81:116-126. doi: 10.1016/j.neuro.2020.09.037. Epub 2020 Sep 28.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Calcium / metabolism*
  • Calcium Signaling*
  • Cell Death / drug effects
  • Cell Line
  • Excitatory Amino Acid Antagonists / pharmacology
  • Humans
  • Induced Pluripotent Stem Cells / drug effects*
  • Induced Pluripotent Stem Cells / metabolism
  • Induced Pluripotent Stem Cells / pathology
  • Methylmercury Compounds / toxicity*
  • Motor Neurons / drug effects*
  • Motor Neurons / metabolism
  • Motor Neurons / pathology
  • Neural Stem Cells / drug effects*
  • Neural Stem Cells / metabolism
  • Neural Stem Cells / pathology
  • Receptors, AMPA / antagonists & inhibitors
  • Receptors, AMPA / metabolism*

Substances

  • Excitatory Amino Acid Antagonists
  • Methylmercury Compounds
  • Receptors, AMPA
  • methylmercuric chloride
  • Calcium