Memantine, a Low-Affinity NMDA Receptor Antagonist, Protects against Methylmercury-Induced Cytotoxicity of Rat Primary Cultured Cortical Neurons, Involvement of Ca2+ Dyshomeostasis Antagonism, and Indirect Antioxidation Effects

Abstract

Methylmercury (MeHg) is an extremely dangerous environmental pollutant that induces severe toxic effects in the central nervous system. Neuronal damage plays critical roles mediating MeHg-induced loss of brain function and neurotoxicity. The molecular mechanisms of MeHg neurotoxicity are incompletely understood. The objective of the study is to explore mechanisms that contribute to MeHg-induced neurocyte injuries focusing on neuronal Ca dyshomeostasis and alteration of N-methyl-D-aspartate receptors (NMDARs) expression, as well as oxidative stress in primary cultured cortical neurons. In addition, the neuroprotective effects of memantine against MeHg cytotoxicity were also investigated. The cortical neurons were exposed to 0, 0.01, 0.1, 1, or 2 μM methylmercury chloride (MeHgCl) for 0.5-12 h, or pre-treated with 2.5, 5, 10, or 20 μM memantine for 0.5-6 h, respectively; cell viability and LDH release were then quantified. For further experiments, 2.5, 5, and 10 μM of memantine pre-treatment for 3 h followed by 1 μM MeHgCl for 6 h were performed for evaluation of neuronal injuries, specifically addressing apoptosis; intracellular free Ca concentrations; ATPase activities; calpain activities; expressions of NMDAR subunits (NR1, NR2A, NR2B); NPSH levels; and ROS formation. Exposure of MeHgCl resulted in toxicity of cortical neurons, which were shown as a loss of cell viability, high levels of LDH release, morphological changes, and cell apoptosis. Moreover, intracellular Ca dyshomeostasis, ATPase activities inhibition, calpain activities, and NMDARs expression alteration were observed with 1 μM MeHgCl administration. Last but not least, NPSH depletion and reactive oxygen species (ROS) overproduction showed an obvious oxidative stress in neurons. However, memantine pre-treatment dose-dependently antagonized MeHg-induced neuronal toxic effects, apoptosis, Ca dyshomeostasis, NMDARs expression alteration, and oxidative stress. In conclusion, the cytoprotective effects of memantine against MeHg appeared to be mediated not only via its NMDAR binding properties and Ca homeostasis maintenance but also by indirect antioxidation effects.

The MARCKS protein amount is differently regulated by calpain during toxic effects of methylmercury between SH-SY5Y and EA.hy926 cells

“…We have previously reported that a decrease in MARCKS amount plays an important role in the MeHg cytotoxicity in both SH-SY5Y and EA.hy926 cells. Furthermore, it is known that MARCKS is cleaved by activated calpain in vitro [ 7 , 21 , 31 , 38 ]. Therefore, we determined the effect of calpain inhibitors on the decrease in MARCKS amount induced by MeHg.…”

“…In our previous studies, amount of the full-length MARCKS protein was significantly decreased by treatment with MeHg, and this protein seems to play a key role in the MeHg toxicity [ 34 , 39 ]. In addition, the involvement of calpain in the MeHg toxicity had been suggested [ 3 , 7 , 21 , 31 , 38 , 41 , 44 ]. Hence, it is important to identify the precise mechanisms behind the regulation of MARCKS levels by MeHg exposure.…”

“…The well-studied calpain isoforms, calpain 1 ( µ -calpain) and calpain 2 (m-calpain), are ubiquitously expressed and regulate important functions of neuronal [ 2 ] and endothelial cells [ 12 ]. MeHg induces calpain activation, which is involved in MeHg cytotoxicity in vitro [ 7 , 21 , 31 , 38 ] and in vivo [ 3 , 41 , 44 ]. Furthermore, regulation of MARCKS function by calpain proteolytic cleavage has been suggested [ 9 , 19 , 36 ].…”

Memantine‐Loaded PEGylated Biodegradable Nanoparticles for the Treatment of Glaucoma

“…Cell viability was assayed with Alamar Blue (Alfagene, Invitrogene, Portugal) at 24 and 48 h as was previously described . Data were analyzed by calculating cell viability through the percentage of Alamar blue reduction compared to the control (untreated cells) …”

Prenatal Deltamethrin Exposure-Induced Cognitive Impairment in Offspring Is Ameliorated by Memantine Through NMDAR/BDNF Signaling in Hippocampus

“…Memantine, a non-competitive, partial NMDAR antagonist, inhibits NMDA glutamate receptors to regulate the glutamatergic system and relieve cognitive and memory deficits in several diseases, such as Alzheimer’s disease, Parkinson’s disease, and ischemia ( Olivares et al, 2012 ; Wang et al, 2016 ). Previous studies have suggested that memantine prevents excitotoxicity induced by Ca 2+ overload in neurons via NMDARs and then upregulates BDNF, thereby enhancing cognitive ability ( Olivares et al, 2012 ; Ranju et al, 2015 ; Liu et al, 2016 ). The key to the therapeutic action of memantine lies in its uncompetitive binding to NMDARs, through which the low affinity and fast dissociation rate kinetics of memantine maintain the physiological function of the NMDA receptor ( Olivares et al, 2012 ).…”