Upstream eventOxidative Stress
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Binding of electrophilic chemicals to SH(thiol)-group of proteins and /or to seleno-proteins involved in protection against oxidative stress during brain development leads to impairment of learning and memory||adjacent||Low||Low|
Life Stage Applicability
|All life stages|
Key Event Relationship Description
In the central nervous system (CNS), glutamate (Glu) is rapidly taken up at the synaptic cleft to mitigate potential excitotoxicity (Meldrum, 2000). Reuptake is carried out by the electrochemical gradient of Glu across the plasma membrane and is accomplished by Glu transporter proteins, referred to as excitatory amino acid transporters (EAATs). These transporter proteins are predominantly expressed in astrocytes, but they are also be found in other neural cells, such as oligodendrocyte, neuron, and microglia membranes (Danbolt, 2001). Functional Glu transporters are located on cell surface membranes. The activities of these transporters are regulated by a redistribution of these proteins to or from the plasma membrane (Robinson 2002), under the control of several signaling pathways. Five different families of EAATs have been recognized (EAAT1–EAAT5). They vary in Na+ and/or K+ coupling abilities. Their names differ based on the presence of the transporter in human or in other mammals (see Table 1).
Astrocyte, oligodendrocyte, microglia
Neuron (somatodendritic), astrocyte (low)
Purkinje cell, astrocyte
Müller cell (retina)
Table 1: Glu transporters in human and mammals and their occurrence in CNS cells. From Rajda et al., 2017
These transporters co-localize with, form physical (co-immunoprecipitable) interactions with, and functionally couple to various 'energy-generating' systems, including the Na(+)/K(+)-ATPase, the Na+/Ca2+ exchanger, glycogen metabolizing enzymes, glycolytic enzymes, and mitochondria/mitochondrial proteins. This functional coupling is bi-directional with many of these systems both being regulated by glutamate transport and providing the 'fuel' to support glutamate uptake (Robinson and Jackson, 2016). The Na+ gradient, which depends on Na/K ATPase pump and consequently of ATP production and intracellular levels, provides the energy to move Glu from the outside into the cells, accompanied by two Na+ and an H+ ; at the same time, K+ moves in the opposite direction (Boron and Boulpaep, 2003). Mitochondrial dysfunction leads to a decrease in ATP synthesis, impaired Ca2+ content, and concomitant increase in the levels of ROS (Reactive Oxygen Species) and RNS (Reactive Nitrogen Species) (Beal, 2005). Free radicals, which are electrically unstable, have a central role in several physiological and pathological processes. Both ROS and RNS originate from endogenous and exogenous sources. Mitochondria, endoplasmic reticulum, peroxisomes, phagocytic cells, and others serve as endogenous sources, and environmental factors, such as alcohol, tobacco, pollution, industrial solvents, pesticides, heavy metals, specified medicines, etc. make up the prepondarance of exogenous factors. Significant amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS,) are formed during oxidative phosphorylation, when the greatest amount of ATP is produced. Cellular antioxidants production serves as a countermeasure against this process (Su et al., 2013; Szalardi et al., 2015). Most cells, including astrocytes, have protective mechanisms against ROS, predominantly in the form of the tripeptide thiol, glutathione (GSH) (Hsie et al., 1996). This process stays in a highly sensitive balance. In the specific case when ROS and RNS synthesis exceeds antioxidant synthesis it results in oxidative stress (Reddy, 2006; Ghafouribar et al., 2008; Su et al., 2013; Szalardi et al., 2015; Valko et al., 2007; Yankovskaya et al., 2003; Senoo-Matsuda et al., 2003; Schon and Manfredi, 2003).
Evidence Supporting this KER
Due to the tight coupling of Glu transporters with energy production, and to the important role of Glu transporters in Glu homeostasis, perturbations of energy metabolism such as mitochondrial dysfunction and increased production of ROS lead to Glu dyshomeostasis (Boron and Boulpaep, 2003). In particular, it was shown that ROS inhibit glutamate uptake by astrocytes (Sorg et al., 1997), and that glutamate release is mediated by ROS-activated volume-sensitive outwardly rectifying anion channels (Liu et al., 2009).
Porciuncula et al., (2003). Methylmercury (2-10 mM) inhibits glutamate uptake in synaptic vesicles isolated from rat brain in a concentration-dependent manner (with LD50 at 50 mM). It also inhibits the H+-ATPase activity in a concentration-dependent manner with similar LD50. This suggests that the vesicular glutamate uptake is impaired by methylmercury and that this effect involves the H+-ATPase.
Roos et al., (2009). Methylmercury induced ROS production in rat brain cortical slices after 2h exposure at 100 and 200 mM and after 5h exposure at 50 mM. Guanosine (0.5 - 5 mM), ebselen (1-5 mM) and diphenyl diselenide (1-5 mM) blocked the methylmercury-induced ROS production. The inhibitor of NMDA receptors, MK801 (50 mM) equally blocked the methylmercury-induced ROS production by two potential mechanisms of action: (i) mercury by affecting mitochondria iincreased ROS formation, which decrease glutamate uptake and consequently increased extracellular glutamate acting on NMDA receptors; (ii) The ROS formation is secondary to overstimulation of NMDA receptors, due to mercury-induced decrease in glutamate uptake.
Roos et al., (2011). Experiments performed in isolated mitochondria from rat liver slices showed that methylmercury (25 mM) increased ROS production (measured by dichlorofluoroscein). Methionine treatment (50-250 mM) was effective in reducing ROS formation.
Juarez et al., (2002) Microdialysis probe in adult Wistar rats showed that acute exposure to methylmercury (10, 100 mM) induced an increase release of extracellular glutamate (9.8 fold at 10 mM and 2.4 fold at 100 mM). This extracellular glutamate level remained elevated at least 90 min following methylmercury exposure.
Allen et al., (2001). Cerebral cortical astrocytes were treated with methylmercury (1 mM for 24h or 10 mM for 30 min) and loaded with [U-13C] glutamate. In the methylmercruy-treated group, a decrease of [U-13C] lactate was observed. This lactate can only be derived from mitochondrial metabolism, via the tricarboxylic acid, showing a link between mitochondrial dysfunction and glutamate metabolism. In addition, the decreased lactate production might be detrimental to surrounding cells, since lactate has been shown to be an important substrate for neurons.
Uncertainties and Inconsistencies
The relationship between oxidative stress associated to mitochondrial dysfunction and glutamate dyshomeostasis is complex and may be bidirectional. Glutamate dysfunction, due to decreased glutamate uptake, can secondarly induce increased ROS production and consequently oxidative stress.
The astrocytic enzyme glutamine synthetase (GS), transforming glutamate in glutamine, which is taken up by neurons, is also a SH-containing protein, which is inhibited by mercury binding (Kwon and Park, 2003). This participate to glutamate dyshomeostasis linking this KE directly to the MIE.
Quantitative Understanding of the Linkage
According to Porciuncula et al., (2003), a decrease of 50% of H+-ATP activity was associated to a decrease of 50% of glutamate uptake following exposure of synaptic vesicles with 5 uM of methylmercury.
Xu et al. (2012) and Feng et al. (2014) observed that in rats treated with 12 umoles/kg for 4 weeks a 4-fold increase in ROS level in cerebral cortex, and a 2-fold increase in protein and DNA peroxidation were associated with about 20% increase of glutamate and 30% decrease of glutamine.
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
In case of glutamate dyshomeostasis, when extracellular concentrations are very high (5 – 10 mM), a mechanism of toxicity called oxidative glutamate toxicity can be observed. It is mediated by an inhibition of cystein uptake leading to a depletion of GSH (Kritis et al., 2015). The GSH depletion decreases the protection against oxidative stress and exacerbates oxidative stress, which, in turn, exacerbates glutamate dyshomeostasis.
Domain of Applicability
Experimental evidences has been observed mainly in rodent, but due to occurrence of oxidative stress and the presence of glutamate in different taxa, it may be much broader, as suggested by similar observations in C. elegans (Wu et al., 2015).
Allen, J. W., H. El-Oqayli, M. Aschner, T. Syversen and U. Sonnewald (2001). "Methylmercury has a selective effect on mitochondria in cultured astrocytes in the presence of [U-(13)C]glutamate." Brain Res 908(2): 149-154.
Bergles DE, Diamond JS, Jahr CE. Clearance of glutamate inside the synapse and beyond. Curr Opin Neurobiol. 1999;9:293–298.
Beal M.F. Mitochondria take center stage in aging and neurodegeneration. Ann. Neurol. 2005;58:495–505.
Berry JD, Shefner JM, Conwit R, Schoenfeld D, Keroack M, Felsenstein D, Krivickas L, David WS, Vriesendorp F, Pestronk A, et al. Design and initial results of a multi-phase randomized trial of ceftriaxone in amyotrophic lateral sclerosis. PLoS One. 2013;8:e61177.
Boron WF, Boulpaep EL. 2003. A cellular and molecular approach. Medical Physiology. Updated Edition. Elsevier, Saunders.
Casado M, Bendahan A, Zafra F, Danbolt NC, Aragón C, Giménez C, Kanner BI. Phosphorylation and modulation of brain glutamate transporters by protein kinase C. J Biol Chem. 1993;268:27313–27317.
Colton CK, Kong Q, Lai L, Zhu MX, Seyb KI, Cuny GD, Xian J, Glicksman MA, Lin C-LG. Identification of translational activators of glial glutamate transporter EAAT2 through cell-based high-throughput screening: an approach to prevent excitotoxicity. J Biomol Screen. 2010;15:653–662.
Danbolt NC. Glutamate uptake. Prog Neurobiol. 2001;65:1–105.
Feng, S., Xu, Z., Liu, W., Li, Y., Deng, Y., Xu, B., 2014. Preventive effects of dextromethorphan on methylmercury-induced glutamate dyshomeostasis and oxidative damage in rat cerebral cortex. Biol Trace Elem Res 159, 332-345.
Fontana AC, Beleboni RO, Wojewodzic MW, Dos SWF, Coutinho-Netto J, Grutle NJ, Watts SD, Danbolt NC, Amara SG. Enhancing glutamate transport: mechanism of action of Parawixin1, a neuroprotective compound from Parawixia bistriata spider venom. Mol Pharmacol. 2007;72:1228–1237.
Ghafourifar P., Mousavizadeh K., Parihar M.S., Nazarewicz R.R., Parihar A., Zenebe W.J. 2008. Mitochondria in multiple sclerosis. Front. Biosci. 13:3116–3126. doi: 10.2741/2913.
Gonzalez MI, Robinson MB. Neurotransmitter transporters: why dance with so many partners? Curr Opin Pharmacol. 2004;4:30–35.
Hediger MA. Glutamate transporters in kidney and brain. Am J Physiol. 1999;277:F487–F492.
Juarez, B. I., M. L. Martinez, M. Montante, L. Dufour, E. Garcia and M. E. Jimenez-Capdeville (2002). "Methylmercury increases glutamate extracellular levels in frontal cortex of awake rats." Neurotoxicol Teratol 24(6): 767-771.
Kritis AA, Stamoula EG, Paniskaki KA, Vavilis TD (2015) Researching glutamate - induced cytotoxicity in different cell lines: a comparative/collective analysis/study. Front Cell Neurosci 9:91 doi:10.3389/fncel.2015.00091
Kwon, O.-S., Park, Y.-J. In vitro and in vivo dose-dependent inhibition of methylmercury on glutamine synthetase in the brain of different species (2003) Environmental Toxicology and Pharmacology, 14 (1-2), pp. 17-24.
Liu HT, Akita T, Shimizu T, Sabirov RZ, Okada Y. 2009. Bradykinin-induced astrocyte–neuron signalling: glutamate release is mediated by ROS-activated volume-sensitive outwardly rectifying anion channels. J Physiol. 587(Pt 10): 2197–2209.
Meldrum BS. 2000. Glutamate as a neurotransmitter in the brain: review of physiology and pathology. J. Nutr. 130(4S Suppl):1007S–1015S.
Mitrovic AD, Plesko F, Vandenberg RJ. Zn2+inhibits the anion conductance of the glutamate transporter EAAT4. J Biol Chem. 2001;276:26071–26076.
Porciuncula, L. O., J. B. Rocha, R. G. Tavares, G. Ghisleni, M. Reis and D. O. Souza (2003). "Methylmercury inhibits glutamate uptake by synaptic vesicles from rat brain." Neuroreport 14(4): 577-580.
Rajda C, Pukoli D, Bende Z, Majalath Z, Vécsei L. 2017. Excitotoxins, Mitochondrial and Redox Disturbances in Multiple Sclerosis. Int J Mol Sci. 2017 Feb; 18(2): 353. doi: 10.3390/ijms18020353.
Reddy P.H. 2006. Mitochondrial oxidative damage in aging and Alzheimer’s disease: Implications for mitochondrially targeted antioxidant therapeutics. J. Biomed. Biotechnol. doi: 10.1155/JBB/2006/31372.
Robinson MB. 2002. Regulated trafficking of neurotransmitter transporters: common notes but different melodies. J Neurochem. 80(1):1-11.
Robinson, M. B. and J. G. Jackson (2016). "Astroglial glutamate transporters coordinate excitatory signaling and brain energetics." Neurochem Int 98: 56-71.
Roos, D. H., R. L. Puntel, M. M. Santos, D. O. Souza, M. Farina, C. W. Nogueira, M. Aschner, M. E. Burger, N. B. Barbosa and J. B. Rocha (2009). "Guanosine and synthetic organoselenium compounds modulate methylmercury-induced oxidative stress in rat brain cortical slices: involvement of oxidative stress and glutamatergic system." Toxicol In Vitro 23(2): 302-307.
Roos, D. H., R. L. Puntel, M. Farina, M. Aschner, D. Bohrer, J. B. Rocha and N. B. de Vargas Barbosa (2011). "Modulation of methylmercury uptake by methionine: prevention of mitochondrial dysfunction in rat liver slices by a mimicry mechanism." Toxicol Appl Pharmacol 252(1): 28-35.
Rothstein JD, Patel S, Regan MR, Haenggeli C, Huang YH, Bergles DE, Jin L, Dykes Hoberg M, Vidensky S, Chung DS, Toan SV, Bruijn LI, Su ZZ, Gupta P, Fisher PB. Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature. 2005;433:73–77.
Sattler R, Rothstein JD. Regulation and dysregulation of glutamate transporters. Handb Exp Pharmacol. 2006;175:277–303
Seal RP, Amara SG. Excitatory amino acid transporters: a family in flux. Annu Rev Pharmacol Toxicol. 1999;39:431–456.
Senoo-Matsuda N., Hartman P.S., Akatsuka A., Yoshimura S., Ishii N. 2003. A complex II defect affects mitochondrial structure, leading to ced-3- and ced-4-dependent apoptosis and aging. J. Biol. Chem. 278:22031–22036. doi: 10.1074/jbc.M211377200.
Schon E.A., Manfredi G. 2003. Neuronal degeneration and mitochondrial dysfunction. J. Clin Investig. 111:303–312. doi: 10.1172/JCI200317741.
Sorg O, Horn TF, Yu N, Gruol DL, Bloom FE. 1997.Inhibition of astrocyte glutamate uptake by reactive oxygen species: role of antioxidant enzymes.Mol Med. 3(7): 431–440.
Su K., Bourdette D., Forte M. 2013. Mitochondrial dysfunction and neurodegeneration in multiple sclerosis. Front. Physiol. 4:169. doi: 10.3389/fphys.2013.00169.
Szalárdy L., Zádori D., Klivényi P., Toldi J., Vécsei L. 2015. Electron transport disturbances and neurodegeneration: From albert Szent-Györgyi’s concept (Szeged) till novel approaches to boost mitochondrial bioenergetics. Oxid. Med. Cell. Longev. 2015:498401. doi: 10.1155/2015/498401.
Valko M., Leibfritz D., Moncol J., Cronin M.T., Mazur M., Telser J. 2007. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 39:44–84. doi: 10.1016/j.biocel.2006.07.001.
Vandenberg RJ, Mitrovic AD, Johnston GA. Molecular basis for differential inhibition of glutamate transporter subtypes by zinc ions. Mol Pharmacol. 1998;54:189–196.
Vandenberg RJ, Ryan RM. Mechanisms of glutamate transport. Physiol Rev. 2013;93:1621–1657.
Wu T, He K, Zhan Q, et al. (2015) MPA-capped CdTe quantum dots exposure causes neurotoxic effects in nematode Caenorhabditis elegans by affecting the transporters and receptors of glutamate, serotonin and dopamine at the genetic level, or by increasing ROS, or both. Nanoscale 7(48):20460-73 doi:10.1039/c5nr05914c
Xing X, Chang L-C, Kong Q, Colton CK, Lai L, Glicksman MA, Lin C-LG, Cuny GD. Structure-activity relationship study of pyridazine derivatives as glutamate transporter EAAT2 activators. Bioorg Med Chem Lett. 2011;21:5774–5777.
Xu, B., Xu, Z.F., Deng, Y., Liu, W., Yang, H.B., Wei, Y.G., 2012. Protective effects of MK-801 on methylmercury-induced neuronal injury in rat cerebral cortex: involvement of oxidative stress and glutamate metabolism dysfunction. Toxicology 300, 112-120.
Yankovskaya V., Horsefield R., Törnroth S., Luna-Chavez C., Miyoshi H., Léger C., Byrne B., Cecchini G., Iwata S. 2003. Architecture of succinate dehydrogenase and reactive oxygen species generation. Science.299:700–704. doi: 10.1126/science.1079605.