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||Moderate||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, ODC, microglia
Neuron (somatodendritic), astrocyte (low)
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 and RNS (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
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).
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