Methylmercury Decreases AMPA Receptor Subunit GluA2 Levels in Cultured Rat Cortical Neurons

Abstract

Methylmercury (MeHg) is a well-known environmental pollutant that has harmful effects on the central nervous systems of humans and animals. The molecular mechanisms of MeHg-induced neurotoxicity at low concentrations are not fully understood. Here, we investigated the effects of low-concentration MeHg on the cell viability, Ca²⁺ homeostasis, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA2 levels, which determine Ca²⁺ permeability of AMPA receptors, in rat primary cortical neurons. Exposure of cortical neurons to 100 and 300 nM MeHg for 7 d resulted in a decrease in GluA2 levels, an increase in basal intracellular Ca²⁺ concentration, increased phosphorylation levels of extracellular signal-regulated kinase (ERK)1/2 and p38, and decreased cell viability. Moreover, glutamate stimulation exacerbated the decrease in cell viability and increased intracellular Ca²⁺ levels in MeHg-treated neurons compared to control neurons. MeHg-induced neuronal cell death was ameliorated by 1-naphthyl acetyl spermine, a specific antagonist of Ca²⁺-permeable, GluA2-lacking AMPA receptors. Our findings raise the possibility that decreased neuronal GluA2 levels and the subsequent increase in intracellular Ca²⁺ concentration may contribute to MeHg-induced neurotoxicity.

INTRODUCTION

Mercury (Hg) is widely used in many commercial products. Among Hg compounds, methylmercury (MeHg) is a well-known environmental contaminant that has adverse effects on humans and animals. MeHg is also produced by the biomethylation of inorganic Hg catalyzed by aquatic microorganisms in aquatic sediments.¹⁾ MeHg accumulates in the aquatic food chain and reaches high concentrations in predators at the top of the marine food pyramid such as tuna, swordfish, tilefish, and whale. Thus, consumption of these sea foods is the main route of human exposure.²⁾ After being absorbed into the body, MeHg is easily distributed into the central nervous system (CNS) by crossing the blood-brain barrier as a complex with L-cysteine through the neutral amino acid transport system.³⁾ It reaches levels that are 3–6 times higher in the brain than in the blood.⁴⁾ Therefore, the CNS is a major target for MeHg-induced toxicity.

High levels of exposure to MeHg during the fetal period induced neurological symptoms of fetal-type Minamata disease, severe MeHg poisoning occurred in the 1950 s and the 1960 s around Minamata Bay in Japan, presenting as decreased IQ, impaired movement, and visuospatial perception exposed, while no or mild symptoms were observed in the mothers.^5–8) Although the effects of MeHg on the developing brain are a major concern, it is important to examine the neurotoxic effects of MeHg on not only the developing brain but also the adult brain because of the risk of MeHg exposure over the life span through contaminated sea foods. Several mechanisms of MeHg-induced neurotoxicity have been reported including mitochondria-dependent cell death,⁹⁾ disruption of redox status and oxidative stress,¹⁰⁾ inhibition of microtubule polymerization,¹¹⁾ inhibition of neuronal differentiation,¹²⁾ interaction with sulfhydryl groups,¹³⁾ and induction of glutamate excitotoxicity.¹⁴⁾ Moreover, some studies have reported the effects of MeHg on neuronal progenitor cells (NPCs) at low-concentration (nanomolar level).^15,16) However, the neurotoxic mechanisms of low-level of MeHg are not fully understood.

Glutamate, an excitatory amino acid in the CNS, plays important roles in neuronal growth, maturation, and synaptic plasticity. Glutamate receptors are mainly divided into two classes based on pharmacology and structural homology: N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors¹⁷⁾; these subtypes play important roles in excitatory synapses. However, overactivation of glutamate receptors triggers cytosolic Ca²⁺ overload and finally induces neuronal death, which is thought to play an important role in neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease.^18–20) Several studies have reported the contribution of NMDA receptors to MeHg-induced neurotoxicity. MeHg-induced neurodegeneration was ameliorated by administration of MK-801 (an NMDA receptor antagonist) in the developing rat brain¹⁴⁾ and human SH-SY5Y neuroblastoma cells.²¹⁾ Moreover, MeHg activates NMDA receptors and induces an increase in dopamine release in the rat brain.²²⁾ In contrast, AMPA receptors have been implicated in the neurotoxicity of MeHg in the peripheral nervous system (PNS). For example, exposure to a chronic low dose (1 or 3 ppm in drinking water) of MeHg accelerated the onset of amyotrophic lateral sclerosis (ALS)-like symptoms such as rotarod failure in ALS model mice.²³⁾ Along with the impairment of motor function, MeHg increased Ca²⁺ concentrations in motor neurons of the model mice, which was ameliorated by both 6-cyano-7-nitroquinoxaline-2,3-dione (a nonselective AMPA receptor antagonist) and 1-naphthyl acetyl spermine (NAS, a specific Ca²⁺-permeable AMPA receptor antagonist). These findings indicated that the motor neurons in the PNS of MeHg-exposed mice have a greater Ca²⁺-permeable AMPA receptor contribution. However, how the functional alterations of AMPA receptors occur, and whether these effects are also induced in the CNS are not known. AMPA receptors are heterotetramers consisting of four subunits, GluA1, GluA2, GluA3, and GluA4, which are encoded by distinct genes and expressed in both neurons and glia in the CNS.^24,25) The Ca²⁺ permeability of the AMPA receptor is dependent on the GluA2 subunit: GluA2 inhibits Ca²⁺ influx into the cytoplasm at steady state, and neurons that contain GluA2-lacking AMPA receptors show high Ca²⁺ permeability and are vulnerable to excitotoxicity.²⁶⁾ GluA2 knockdown in rat brain results in neuronal cell death and degeneration, which is increased by global ischemia,²⁷⁾ suggesting that the decrease in GluA2 levels makes neurons vulnerable to excitotoxicity. Therefore, we hypothesized that MeHg exposure decreases neuronal GluA2 levels and induces functional alterations in AMPA receptors, which leads to disruption of neuronal Ca²⁺ homeostasis and neuronal death. Moreover, some studies have shown the neurotoxicity of relatively high concentrations (micromolar levels) of MeHg in cultured neurons.^9,11,21) In the current study, we examined the mechanism of MeHg-induced neuronal damage, focusing on AMPA receptor-mediated excitotoxicity in rat primary cortical neurons at relatively low concentrations (nanomolar levels).

MATERIALS AND METHODS

Materials

Methylmercury chloride, glycerol, and MgSO₄ were purchased from Kanto Chemical (Tokyo, Japan). Cytosine arabinofuranoside (Ara-C), L-glutamine, and MgCl₂ were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Sodium-L-glutamate, D-(+)-glucose, KCl, bromophenol blue (BPB), 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), KH₂PO₄, glycine, deoxycholate (DOC), 2-mercaptoethanol, proteasome inhibitor cocktail, sodium dodecyl sulfate (SDS), Nonidet P-40, Tris base, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Nacalai Tesque (Kyoto, Japan). NAS was purchased from Cayman Chemical (Ann Arbor, MI, U.S.A.). Streptavidin agarose ultra-performance was purchased from Cosmo Bio (Tokyo, Japan). Fluo-8 AM was purchased from AAT Bioquest (Sunnyvale, CA, U.S.A.). Tributyltin chloride, dimethyl sulfoxide (DMSO), NaCl, CaCl₂, and sodium orthovanadate were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). 1,2-Bis(2-aminophenoxy)ethane-N,N’,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester, BAPTA/AM) was purchased from Adooq Bioscience (Irvine, CA, U.S.A.). Sulfo-NHS-LC-biotin was purchased from Thermo Fisher Scientific (Waltham, MA, U.S.A.).

Cell Culture

The study was approved by the animal ethics committee of Hiroshima University. The following procedures were performed under sterile conditions. Primary neuronal culture was performed as previously described²⁸⁾ with some modifications. The prefrontal part of the cerebral cortex was dissected from fetal Slc:Wistar/ST rats at gestation day 18, and the cells were dissociated by gentle pipetting and plated onto culture plates at a density of 3 × 10⁵ cells/cm². The cultures were incubated in Neurobasal™ medium (Life Technologies, Carlsbad, CA, U.S.A.) supplemented with B-27 supplement (Life Technologies), 0.5 mM L-glutamine, and antibiotics (penicillin G and streptomycin sulfate; Meiji Seika, Tokyo, Japan). The cultures were maintained at 37 °C in an atmosphere of humidified 5% CO₂ in air for 7 d from day in vitro (DIV) 0 to DIV 8. Culture medium was replaced at DIV 1 and 4, and 10 µM Ara-C was added to inhibit the proliferation of non-neuronal cells at DIV 1. Neurons were exposed to MeHg, tributyltin (TBT), or DMSO (control) after every medium change from DIV 1 to DIV 8 for 7 d. 5 µM NAS was added from DIV 6 to DIV 8 and 10 or 30 µM BAPTA-AM for the last 3 h at DIV 8.

Cell Viability Assay

Cell viability was measured using the water-soluble tetrazolium salt (WST)-1 assay as previously described.²⁹⁾ Briefly, the cell culture medium was replaced with WST-1 reagent [5 mM 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazorium, monosodium salt (Dojindo, Kumamoto, Japan) and 0.2 mM 1-methoxy-5-methylphenazinium methyl sulfate (Dojindo) in phosphate-buffered saline (PBS)] diluted 1 : 12 in cell culture medium. After incubation for 2 h at 37 °C in a humidified 5% CO₂ incubator, absorbance of the converted dye was measured at 415 nm and cell viability was calculated.

Measurement of Intracellular Ca²⁺ Concentrations

Ca²⁺ concentration was measured as previously described³⁰⁾ with some modifications. Rat primary cortical neurons on a 35 mm glass base dish (IWAKI, Tokyo, Japan) were loaded with 5 µM Fluo-8 AM for 30 min in HEPES-buffered salt solution (HBSS) containing 125 mM NaCl, 5 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 1.2 mM CaCl₂, 6 mM glucose, and 25 mM HEPES. The slides were washed with HBSS, and the changes in intracellular Ca²⁺ concentration were evaluated with a live imaging system, LCV110 (OLYMPUS, Tokyo, Japan), by measuring the fluorescence intensity at 514 nm. Glutamate (25 µM) or vehicle (H₂O) was added to neurons 1 min after the start of measurement. Each point represents the mean of 20 cells.

Whole Cell Protein Isolation

Whole cell protein isolation was performed as previously described.³¹⁾ Briefly, the cells were washed with PBS buffer and lysed in lysis buffer containing 50 mM Tris–HCl, 1% nonidet P-40, 20 mM ethylenediaminetetraacetic acid (EDTA), protease inhibitor cocktail (1 : 100), 1 mM sodium orthovanadate, and 1 mM PMSF. The mixture was rotated at 4 °C and centrifuged at 13500 rpm for 20 min, after which the supernatant was transferred to a microtube.

Membrane Protein Isolation

After the cells were washed with PBS containing 1 mM CaCl₂ and 1.3 mM MgCl₂ (PBS (+)), they were incubated with 1 mg/mL sulfo-NHS-LC-biotin in PBS (+) for 30 min at 4 °C to bind cell surface proteins. Then, PBS (+) containing 100 mM glycine was added to the culture dish to stop the biotinylation reaction, and the cells were lysed in lysis buffer containing 0.5% DOC, 0.5% Nonidet P-40, 0.2% SDS, 1% protease inhibitor cocktail, 1 mM sodium orthovanadate, and 1 mM PMSF in PBS (+). The lysates were centrifuged at 13500 rpm for 20 min at 4 °C. The supernatant fraction was incubated overnight at 4 °C with streptavidin agarose beads (Sigma-Aldrich) to promote the binding between the streptavidin beads and the biotin-conjugated surface proteins. The beads were centrifuged at 500 rpm for 5 min at 4 °C. Next, the supernatant was tested for the presence of intracellular proteins. The beads bound to surface proteins were washed with lysis buffer before being resuspended in denaturing buffer (100 mM Tris–HCl, 4% SDS, 20% glycerol, 0.004% BPB, and 5% mercaptoethanol) and boiled at 95 °C for 10 min. The sample was then analyzed by Western blotting.

Western Blotting

Western blotting was performed as previously described³²⁾ with some modifications. The denaturing buffer was added to the protein extracts, and the extracts were incubated at 95 °C for 3 min. The extracts were then separated by electrophoresis, and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% skim_milk for 1 h, and then incubated with the primary antibody overnight, before being incubated with the secondary antibody overnight. The proteins were detected using Chemi-Lumi One L (Nacalai Tesque) or Chemi-Lumi One Ultra (Nacalai Tesque). Chemiluminescence signals were detected using ImageQuant LAS 4000 (GE Healthcare, Chicago, IL, U.S.A.) and quantified using digital imaging software (Image J; NIH, Bethesda, MD, U.S.A.). The primary antibodies used for Western blotting were anti-GluA1 (MAB2263, Millipore, Billerica, MA, U.S.A.), anti-GluA2 (MAB397, Millipore), anti-GluA3 (#4676, Cell Signaling Technology, Danvers, MA, U.S.A.), anti-GluA4 (#8070, Cell Signaling Technology), anti-phospho-extracellular signal-regulated kinase (ERK)1/2 (#9101, Cell Signaling Technology), anti-ERK1/2 (#9102, Cell Signaling Technology), anti-phospho-p38 (#4511, Cell Signaling Technology), anti-p38 (#8690, Cell Signaling Technology), anti-phospho-c-Jun N-terminal kinase (JNK) (#9251, Cell Signaling Technology), anti-JNK (#9258, Cell Signaling Technology), anti-β-tubulin (014-25041, FUJIFILM Wako), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (G9545, Sigma-Aldrich) antibodies.

Statistical Analysis

All experiments were replicated at least three times, and representative data are shown. Data are expressed as the mean ± standard error of the mean (S.E.M.). Statistical analyses of the data were performed using one-way ANOVA followed by Tukey’s test and Dunnett’s test for multiple comparisons, or Student’s t-test for two independent samples. p < 0.05 was considered statistically significant.

RESULTS

Exacerbation of Glutamate-Induced Excitotoxicity in MeHg-Treated Cortical Neurons

Although studies have evaluated the effects of MeHg exposure for 3–48 h at relatively high concentration on neuronal cells,^11,21,33,34) there are little evidence of the effects of low-levels MeHg exposure for several days. Hence, rat primary cortical neurons were exposed to various concentrations of MeHg for 7 d, and the cell viability was evaluated by WST-1 assay. Cell viability decreased in a dose-dependent manner after exposure to MeHg, reaching statistical significance at 100 nM (Fig. 1a). Next, we investigated the sensitivity of MeHg-treated neurons to glutamate. After 7 d of exposure to DMSO (control), 100, and 300 nM MeHg, or 50 nM tributyltin (TBT, positive control, which is known to increase neuronal sensitivity to glutamate),³⁵⁾ neuronal viability was measured after treatment for 1 h with vehicle or 25 µM glutamate, a concentration that partially induces neuronal death. After vehicle treatment, cell viability was not changed between control and TBT-exposed neurons, and that in MeHg-exposed neurons was moderately but significantly decreased to 83 and 58% (100 and 300 nM MeHg, respectively) compared to control neurons (100%) (Fig. 1b, left), which coincides with Fig. 1a. After glutamate treatment, the viability of TBT-treated neurons was significantly decreased, and that of 100- and 300-nM-MeHg-treated neurons was markedly decreased compared to the viability of control neurons (Fig. 1b, right). These results indicate that MeHg exposure exacerbated glutamate-induced excitotoxicity in cortical neurons.

Fig. 1. Glutamate Excitotoxicity Induced by Methylmercury (MeHg) Exposure in Primary Neurons

(a) Rat primary cortical neurons were exposed to dimethyl sulfoxide (DMSO, Cont.) and 30–1000 nM MeHg for 7 d, and then cell viability was measured by WST-1 assay. (b) Rat primary cortical neurons were exposed to DMSO, 50 nM tributyltin (TBT, positive control), and 100 and 300 nM MeHg for 7 d and then to vehicle (H₂O) or 25 µM glutamate (Glu.) for 1 h. Cell viability was measured using the WST-1 assay. Data are expressed as the mean ± S.E.M. (n = 3). ** p < 0.01 vs. Cont. WST-1, water-soluble tetrazolium-1 salt.

Excitotoxicity is triggered by excessive Ca²⁺ entry into cells; therefore, MeHg-treated neurons could be expected to have increased Ca²⁺ influx upon stimulation by glutamate. To investigate the effects of MeHg on glutamate-induced Ca²⁺ influx, rat primary cortical neurons were exposed for 7 d to DMSO (control), 100 nM MeHg, and 50 nM TBT (positive control, which is known to increase glutamate-induced Ca²⁺ influx)³⁵⁾ and treated with 25 µM glutamate. Intracellular Ca²⁺ concentration was measured as fluorescence using a Fluo8-AM probe. The average intensity was markedly increased after glutamate stimulation in control, MeHg-exposed, and TBT-exposed neurons (Figs. 2a, b), with the increase in the average intensity being greater in MeHg- or TBT-exposed neurons than in control neurons. The area under the curve (AUC) of the average intensity showed that MeHg and TBT significantly increased glutamate-induced Ca²⁺ influx to 234 and 259% of the control, respectively (Fig. 2c). In addition, we measured basal Ca²⁺ levels in neurons and demonstrated that intracellular Ca²⁺ concentrations were higher in neurons exposed to 100 and 300 nM MeHg and 50 nM TBT than in control neurons (Fig. 2d). These results indicate that MeHg exposure increased Ca²⁺ entry into neurons under basal conditions and after glutamate stimulation.

Fig. 2. Effects of MeHg Exposure on Ca²⁺ Influx in Primary Neurons

Rat primary cortical neurons were exposed to dimethyl sulfoxide (DMSO, Cont.), 50 nM tributyltin (TBT, positive control), and 100 nM MeHg for 7 d and then with 25 µM glutamate (Glu.) was treated. Intracellular Ca²⁺ concentrations were measured as fluorescence using a Fluo8-AM probe. (a) Fluorescence images of neurons at 0, 5, 60, and 300 s after glutamate stimulation. Scale bar: 80 µm. (b) The average fluorescence intensity from 0 to 6 min was quantified. Application of 25 µM glutamate to neurons at 1 min is indicated by upward arrow. (c) The area under the curve of glutamate-induced Ca²⁺ influx was calculated. Data are expressed as the mean ± S.E.M. (n = 20). ** p < 0.01 vs. Cont. (d) Rat primary cortical neurons were treated for 7 d. Basal Ca²⁺ concentrations were measured for 6 s as fluorescence using a Fluo8-AM probe.

Effects of MeHg on AMPA Receptor Subunit Levels in Primary Cortical Neurons

Glutamate excitotoxicity is caused by the activation of glutamate receptors, and the composition of AMPA receptor subunits affects Ca²⁺ influx into neurons. To investigate the effects of MeHg on AMPA receptor subunit levels, rat primary cortical neurons were exposed to 30, 100, and 300 nM MeHg for 7 d, and the protein levels of the AMPA subunits were analyzed by Western blotting (Figs. 3a, b). GluA2 levels decreased to 84, 72, and 61% of the control in neurons exposed to 30, 100, and 300 nM MeHg, respectively; the decrease reached significance with 100 and 300 nM MeHg (Figs. 3a, b). In contrast, the protein levels of GluA1, GluA3, and GluA4 did not change following MeHg exposure (Figs. 3a, b). These results indicate that exposure to MeHg specifically decreases GluA2 levels without affecting the levels of other subunits.

Fig. 3. Effects of MeHg on Protein Levels of AMPA Receptor Subunits in Primary Neurons

Rat primary cortical neurons were exposed to dimethyl sulfoxide (DMSO, Cont.), 30, 100, and 300 nM MeHg for 7 d. (a) Protein levels of GluA1, GluA2, GluA3, GluA4, and β-tubulin were determined by Western blotting. (b) Protein levels were quantitatively analyzed using ImageJ software and normalized to β-tubulin protein levels. Data are expressed as mean ± S.E.M. (n = 4) * p < 0.05, ** p < 0.01 vs. Cont. (c) After exposure to DMSO, 100 and 300 nM MeHg for 7 d, cell surface proteins were biotinylated and separated from intracellular proteins. GluA2 protein levels were determined by Western blotting. N-Cadherin (N-cad) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as cell surface markers and cytosol markers, respectively. (d) Cortical neurons were exposed to DMSO, and 100 nM MeHg from day in vitro (DIV) 1 to DIV 8, with or without 5 µM 1-naphthyl acetyl spermine (NAS) from DIV 6 to DIV 8. Cell viability was measured by the water-soluble tetrazolium-1 assay. Data are expressed as the mean ± S.E.M. (n = 3) ** p < 0.01.

As functional AMPA receptors are expressed on plasma membranes, we measured GluA2 levels in the membrane fraction and found that membrane GluA2 levels (cell surface fraction) decreased after 7-d exposure to 100 and 300 nM MeHg (Fig. 3c). Next, to investigate the involvement of the decrease in GluA2 in MeHg-induced neuronal cell death, we examined the viability of rat cortical neurons exposed to 100 nM MeHg for 7 d, with or without NAS, a specific inhibitor of Ca²⁺-permeable, GluA2-lacking AMPA receptors. Treatment with NAS significantly attenuated the decrease in cell viability caused by 100 nM MeHg (Fig. 3d). These results suggest that the MeHg-induced decrease in viability could be attributed to the MeHg-induced decrease in GluA2 levels.

Effect of MeHg Exposure on Mitogen-Activated Protein Kinases

Mitogen-activated protein kinases (MAPKs) are a conserved family of Ca²⁺-dependent enzymes that regulate fundamental intracellular processes including growth, proliferation, differentiation, and apoptosis.³⁴⁾ To investigate the effects of MeHg on intracellular Ca²⁺ levels and MAPK activity, rat cortical neurons were exposed to 100 and 300 nM of MeHg for 7 d, and the phosphorylation levels of three major groups of MAPKs—ERK 1/2, p38 MAPKs (p38), and JNKs—were analyzed by Western blotting (Fig. 4a). After MeHg exposure, p-ERK level tended to be increased to 166% of control at 100 nM, and was significantly increased to 230% of control at 300 nM (Fig. 4b). In addition, the p-p38 level was significantly increased to 162 and 178% of the control by exposure to 100 and 300 nM of MeHg, respectively (Fig. 4c). However, the p-JNK level was not changed by MeHg exposure (Fig. 4d). MeHg-induced phosphorylation of ERK and p38 was abolished by treatment with BAPTA-AM, an intracellular Ca²⁺ chelator (Fig. 4e), indicating that MeHg-induced Ca²⁺ influx induced phosphorylation of ERK and p38.

Fig. 4. Effects of MeHg on Phosphorylation of ERK, p38, and JNK

Rat primary cortical neurons were exposed to dimethyl sulfoxide (DMSO, Cont.), 100 and 300 nM MeHg for 7 d. (a) Levels of phosphorylated or total ERK, p38, and JNK were detected by Western blotting. Phosphorylation levels of (b) ERK, (c) p38, and (d) JNK were quantitatively analyzed using ImageJ software and normalized to total protein levels. Data are expressed as mean ± S.E.M. (n = 3) * p < 0.05, ** p < 0.01 vs. Cont. (e) After exposure to DMSO, 100 and 300 nM MeHg for 7 d, neurons were treated with 10 or 30 µM BAPTA-AM for 3 h, and levels of phosphorylated or total ERK and p38 were detected by Western blotting.

DISCUSSION

In the current study, we examined the effects of MeHg on the cell viability, intracellular Ca²⁺ concentrations and subsequent phosphorylation levels of MAPKs, and GluA2 levels in rat primary cortical neurons. MeHg decreased cell viability in a dose-dependent manner, reaching statistical significance at 100 nM (Fig. 1a). The increase in intracellular basal Ca²⁺ levels in neurons exposed to 100 and 300 nM MeHg negatively correlated with neuronal viability (Fig. 2d). Therefore, we concluded that the neuronal cell death induced by MeHg resulted from an increase in Ca²⁺ influx. Treatment with 100 and 300 nM MeHg decreased GluA2 levels without affecting the levels of other subunits (Fig. 3), which led to a relative increase in GluA2-lacking AMPA receptors. We have previously reported a similar induction of neurotoxicity mediated by decreased GluA2 levels by some environmental chemicals.^36–38) The Ca²⁺ permeability of the AMPA receptor is dependent on the GluA2 subunit, and neurons that contain GluA2-lacking AMPA receptors show high Ca²⁺ permeability and are vulnerable to excitotoxicity.²⁶⁾ Therefore, MeHg-induced neurotoxicity may be attributed to a decrease in GluA2 levels and a subsequent increase in intracellular Ca²⁺ levels. Indeed, like the NAS-mediated amelioration of MeHg-increased Ca²⁺ levels in motor neurons of ALS model mice,²³⁾ we observed partial amelioration of MeHg-induced cell death in cortical neurons by NAS in this study (Fig. 3d). These results indicate the contribution of Ca²⁺-permeable AMPA receptors to MeHg-induced neuronal cell death. Membrane depolarization induced by the activation of AMPA receptors relieves the Mg²⁺ blockade of NMDA receptors, which results in Ca²⁺ influx through NMDA receptors.^39,40) Therefore, the GluA2 decrease may contribute, at least in part, to the NMDA receptor-mediated MeHg neurotoxicity. The protective effects of NAS against MeHg-induced neuronal cell death were partial, possibly because of the involvement of independent mechanisms such as decreased mitochondrial function,⁹⁾ oxidative stress,¹⁰⁾ or AMPA receptor-independent activation of NMDA receptors. In addition, glutamate stimulation exacerbated the decrease in cell viability (Fig. 1b) and increased intracellular Ca²⁺ levels (Figs. 2a–c) in MeHg-treated neurons compared to those in control neurons, suggesting that MeHg increased the susceptibility of neurons to glutamate-induced excitotoxicity. GluA2 knockout mice exhibit a decrease in exploratory behavior and spontaneous locomotor activity and impairment of learning memory.^41–43) Therefore, MeHg-induced decrease in GluA2 levels would also result in these behavioral abnormalities.

MAPK cascades comprise a common, conserved mechanism of signal transduction that links extracellular signals to fundamental intracellular processes and regulates various cellular activities including growth, proliferation, differentiation, and apoptosis.⁴⁴⁾ Here, we demonstrated that 100 and 300 nM MeHg induced phosphorylation of ERK and p38 in neurons (Figs. 4a–c), which was abolished by treatment with BAPTA, an intracellular Ca²⁺ chelator (Fig. 4e). Consistent with our results, studies have reported the activation of MAPKs in MeHg-treated neuronal cells: 750 nM MeHg induced oxidative stress and subsequent activation of the p38 pathway, contributing to a decrease in cell viability in SH-SY5Y cells.⁴⁵⁾ Moreover, exposure to MeHg (4 or 12 µmol/kg) resulted in Ca²⁺ overload, activation of MAPKs including p38, and high levels of caspase 3 and Bax/Bcl2, which finally induced neuronal apoptosis in the cerebral cortex.⁴⁶⁾ Therefore, we inferred that MeHg decreased GluA2 levels and subsequently increased Ca²⁺ influx, which resulted in the disruption of intracellular Ca²⁺ homeostasis or overactivation of Ca²⁺-dependent signals, including the ERK and p38 pathways, and finally induced neuronal cell death. Lead acetate, which has been reported to decrease GluA2 in our previous study, also activated ERK and p38 but not JNK.³⁷⁾ Therefore, MeHg and lead acetate appear to have similar actions in terms of GluA2 decrease and activation of these kinases. The activation of kinases could be attributed to the increase in basal Ca²⁺ levels. Given that the concentration of MeHg investigated in our study was relatively low (nanomolar) compared to other in vitro studies on micromolar MeHg-induced neurotoxicity,^9,11,21) there is a possibility that this decrease in GluA2 levels and increase in Ca²⁺ influx are initial events that contribute to MeHg-induced neurotoxicity.

The GluA2 gene is regulated by a variety of transcription factors such as Sp1, nuclear respiratory factor-1, and RE-1 silencing transcription factor (REST), also known as the neuron-restrictive silencer factor.^47,48) Guida et al. reported that acute exposure of rat cortical neurons to MeHg (1 µM for 12 and 24 h) resulted in a decrease in microRNA 206 and a subsequent increase in REST expression in rat cortical neurons,⁴⁹⁾ although their exposure model was different from our model (100 and 300 nM for 7 d). In addition to transcriptional regulation of the GluA2 gene, increased protein degradation can be related to MeHg-induced decrease in GluA2 levels. Ubiquitination contributes to intracellular trafficking of AMPA receptors to late endosomes, which finally leads to protein degradation of AMPA receptors in lysosomes.⁵⁰⁾ Our preliminary data indicated that the GluA2 mRNA expression was not changed by MeHg exposure (data not shown). Although it is unknown whether MeHg affects the ubiquitination system, MeHg may, at least partly, promote GluA2 protein degradation through changes in the ubiquitination of GluA2.

In 1959, the mercury concentration in fish from Minamata Bay was 15 ppm and in shellfish was 108–178 ppm.⁵¹⁾ The mercury concentrations in the postmortem brain of cases of MeHg poisoning reached approx. 10 ppm (49.8 µM),⁵²⁾ which is higher than the concentration used in our in vitro experiments (100–300 nM). Since 1960 s, in contrast, the mercury concentrations in fishes were gradually decreased, and these were 0.01–1.74 ppm in 1989.⁵¹⁾ In the meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), the acceptable daily intake (ADI) for MeHg of 0.23 µg/kg b.w./d was confirmed.⁵³⁾ The estimated intake of MeHg and Hg in some geographical areas are relatively low, and not exceeding the ADI.⁵⁴⁾ Therefore, although the toxic level of MeHg exposure from the environment in our daily lives is decreasing, exposure can increase with sustained consumption of seafood with high levels of contamination of MeHg, and the MeHg concentration in the human brain can reach the levels studied here (100–300 nM). In fact, the mean MeHg concentrations in the cerebrum of residents living in non-polluted areas with mercury were as follows; 0.009 µg/g (41.7 nM) in 1972–1973, and 0.003 µg/g (13.9 nM) in 1987–1991.⁵⁵⁾ Hence, it is important to understand the risk of MeHg at concentrations investigated in the current study. In conclusion, exposure to MeHg induced a decrease in GluA2 levels in cultured neurons, which may be responsible for increased Ca²⁺ influx and neuronal cell death. Our results raise the possibility that decreased neuronal GluA2 levels contribute to MeHg-induced excitotoxicity.

Acknowledgments

This study was supported by Grants-in-Aid for Scientific Research (#15H02826 and #20H04342 to Y.K.), the Japan Society for the Promotion of Science (JSPS).

Conflict of Interest

The authors declare no conflict of interest.

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