Upstream eventIncreased, Intracellular Calcium overload
N/A, Mitochondrial dysfunction 1
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Binding of agonists to ionotropic glutamate receptors in adult brain causes excitotoxicity that mediates neuronal cell death, contributing to learning and memory impairment.||non-adjacent||High|
Life Stage Applicability
Key Event Relationship Description
One of the mitochondrial functions is to buffer intracellular Ca2+ levels facilitating the maintenance of Ca2+ homeostasis in the cell. In the case of Ca2+ overload, mitochondria are not able to buffer the excess of Ca2+ that leads to mitochondrial dysfunction measured by the increased generation of reactive oxygen species (ROS), triggering mitochondrial permeability transition pore opening (Choi et al.,2013) and reduced ATP production (reviewed in Gleichmann and Mattson, 2011).
Evidence Supporting this KER
There is functional and structural mechanistic understanding supporting the relationship between KE "Ca2+ influx, increased" and KE "Mitochondrial dysfunction".
The increase in cytoplasmic Ca2+ can cause the activation of plasma membrane and endoplasmic reticulum (ER) Ca2+-ATPases that results in higher ATP demand. At the same time elevated Ca2+ can cause reduced levels of ATP by the direct uptake of the cation into the matrix that utilizes the proton circuit and directly competes with mitochondrial ATP synthesis (reviewed in Nicholls, 2009).
Ca2+ overload besides of being detrimental to mitochondrial energy production can also induce mitochondrial ROS generation. A number of possible mechanisms have been suggested by which Ca2+ overload can increase ROS production including: 1) stimulated increase of metabolic rate by Ca2+, 2) stimulated nitric oxide production by Ca2+, 3) Ca2+ induced cytochrome c dissociation, 4) Ca2+ induced cardiolipin peroxidation, 5) Ca2+ induced mitochondrial permeability transition pore (MPTP)opening with release of cytochrome c (leading to apoptosome formation and caspase-3 activation)and apoptosis inducing factor (AIF), decreased level of reduced glutathione (GSH), the antioxidative enzymes, and 6) Ca2+-calmodulin dependent protein kinase activation (reviewed in Peng and Jou, 2010; Gleichmann and Mattson, 2011). It is worth mentioning that mitochondrial ROS increase is capable of modulating Ca2+ dynamics causing further increase of Ca2+ levels.
The cytoplasmic and mitochondrial Ca2+ levels, the oxidative stress and the energy production are very closely inter-related. For example, decreased (or lack) of ATP production can affect the function of plasma membrane Ca2+ pump activity causing Ca2+ overload, oxidative stress and further restriction in ATP generating capacity (reviewed in Nicholls, 2009). Prolonged oxidative stimuli cause further mitochondrial dysfunction, including the decrease of mitochondrial transmembrane potential (ΔΨm), further overload of mitochondrial calcium, and opening of mitochondrial permeability transition pore (MPTP) (Choi et al., 2013).
Mitochondria within dendrites are elongated and perform extensive directional and lateral movement at physiological conditions. Under an excitotoxic exposure to glutamate, mitochondrial movement has been found to be inhibited and mitochondria change morphology becoming rounded and swollen. Although blocking mitochondrial ATP production is sufficient to inhibit mitochondrial movement (Rintoul et al., 2003), research has shown that the collapse of mitochondrial structure requires extracellular Ca2+ influx via NMDA receptors (Rintoul et al., 2003; Pivovarova et al., 2004; Shalbuyeva et al., 2006), suggesting that structural, mechanistic understanding is also available supporting this KER.
In neurons, the high mitochondrial content in axons and dendrites closely correlates with the high energy demand in these structures that is needed to pump the ions that underlie the generation of action potentials mediated by the electrochemical gradients (Attwell and Laughlin, 2001).
Include consideration of temporal concordance here
- DomA has been shown to cause a significant time- and concentration-dependent increase of ROS production in mouse cerebellar granule neurons (CGNs) and the maximal effect (2.5 fold increase) has been recorded 1 h after exposure (Giordano et al., 2006). The time course involved the measurement of oxidant-sensitive fluorescent dye DCFH2-DA from 15 to 120 min and the concentrations assessed are 1 and 10 µM DomA (Giordano et al., 2006). ROS production is higher in Gclm (-/-) neurons lacking glutathione (21.97 pmol DCF/mg of protein) than in Gclm (+/+) neurons (10.23 pmol DCF/mg of protein) after treatment with DomA (Giordano et al., 2006). In the same study, treatment of mouse CGNs with 1 and 10 µM DomA elevate intracellular Ca2+ by approximately 5 or 8 fold higher compared to controls, respectively (Giordano et al., 2006), showing that the cytosolic Ca2+ increase (upstream KE) is higher than the down-stream KE (ROS production due to mitochondrial dysfunction).
- The same research group have measured intracellular Ca2+ concentrations at different time points after DomA treatment of cerebellar granule neuons (CGNs) from mice lacking the modifier subunit of glutamate-cysteine ligase (Gclm). Glutamate-cysteine ligase (Glc) catalyzes the first and rate-limiting step in glutathione (GSH) biosynthesis. CGNs from Gclm (-/-) mice have very low levels of GSH and are 10-fold more sensitive to DomA-induced toxicity than CGNs from Gclm (+/+) mice (Giordano et al., 2007). The low DomA dose (0.1μM) causes a small and delayed increase in intracellular Ca2+ concentration with a full recovery by 20 min, whereas, the higher DomA concentration (10 μM) causes a rapid and robust increase in intracellular Ca2+, which lasts even after 25 min, revealing that upstream KE (cytosolic Ca2+) happens much earlier than the down-stream KE (ROS production). Interestingly, in the same study the mitochondrial Ca2+ concentration has been measured and showed that 0.1μM DomA causes an increase by about 3-fold, with a delay of about 15 min, but no changes in mitochondrial Ca2+ concentration have been observed at 10 μM of DomA (Giordano et al., 2007).
- Mice injected intraperitoneally (i.p.) at a dose of 2 mg/kg of DomA once a day for 3 weeks show markedly lowered (1.5-2 fold) respiratory control ratio, mitochondrial ATP production rate, electron transport chain activity and cellular ATP concentration (Lu et al., 2012; Wu et al., 2013). In Lu et al. 2013 the same treatment in mice causes a 3 or 1.8-fold decrease in electron transport chain activity and mitochondrial ATP content, respectively. Western blot analysis demonstrate that the level of complex I-V proteins (mt-Nd6, Sdha, Uqcrc1, mt-Co1, and Atp5a1) in the hippocampus of DomA-treated mice is significantly decreased compared to controls (Lu et al., 2012). In the same study, DomA treatment significantly elevate the expression of NOX subunits (p47phox and gp91phox), of ROS (3.2 fold increase) and protein carbonyl levels, as well as the production of superoxide anion radicals (Lu et al., 2012). Under the same experimental conditions an increase of NOX activity (2 fold) has been reported in the hippocampus of DomA-treated mice (Lu et al., 2013). Furthermore, DomA exposure induces ER stress by increasing the levels of phosphorylated pancreatic endoplasmic reticulum-resident kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α), glucose-regulated protein 78, C/EBP homologous protein (CHOP), X-box binding protein 1 (XBP1) and the phosphorylated inositol-requiring enzyme 1 (IRE1) (Lu et al., 2012).
- DomA (0.75 mg/kg body weight) administered intravenously in adult rats reveals no remarkable changes at the mRNA level of iNOS expression but demonstrates significant induction in the expression of iNOS protein level in the neurons and astrocytes of the hippocampus (Ananth et al., 2003).
|Stressor||Experimental Model||Tested concentrations||Exposure route||Exposure duration||Increased intracelllular Ca 2+ levels (KE up) (measurements, quantitative if available)||Mitochondrial dysfunction (KE down) (measurements, quantitative if available)||References||Temporal Relationship||Dose-response relationship||Incidence||Comments|
|DomA||Mouse cerebellar granule neurons (CGNs) from Gclm (+/+) and Gclm (−/−) mice||0.01 to 10 µM||Time course (15 to 120 min)||5 and 8 fold increase of [Ca2+]i compared to controls.||Increase in ROS production (2.5 fold) after 1 h of exposure.||Giordano et al., 2006||Same dose||Incidence of upstream KE (Increased intracelllular Ca 2+ levels) is higher than the incidence of downstream KE (mitochondrial dysfunction)|
|DomA||CGNs from Gclm (+/+) and Gclm (−/−) mice||0.01 to 10 µM||Time course (0 to 25 min)||0.1μM domoic acid caused a small and delayed increase (4 fold) in [Ca2+]i, with a full recovery by 20 min. In contrast, the higher concentration of domoic acid (10μM) caused a rapid and robust increase (8 fold) in [Ca2+]i, which was still elevated after 25 min. 0.1μM DomA increases [Ca2+]M by about 3 fold, with a delay of about 15 min. In contrast, no changes in [Ca2+]M were observed following 10μM of DomA.||DomA (0.1μM) caused a 3 fold increase in DHR fluorescence, which accumulates in mitochondria and fluoresces when oxidized by ROS or reactive nitrogen species. This occurred between 1 and 2 h and was higher in CGNs from Gclm (−/−) mice.||Giordano et al., 2007||Yes||Same dose|
|DomA||Adult mice||2 mg/kg||intraperitoneally (i.p.)||Once a day for 3 weeks||Decreased respiratory control ratio (1.5-2 fold), mitochondrial ATP production rate, electron transport chain activity, cellular ATP concentration, electron transport chain activity (3 fold) and mitochondrial ATP content (1.8 fold).||Lu et al., 2012; Lu et al. 2013; Wu et al., 2013|
|DomA||Adult rats||0.75 mg/kg||Induction in the expression of iNOS protein level.||Ananth et al., 2003|
Gap of knowledge: there are no studies available showing that Glufosinate (GLF) increases intra-cellular calcium levels causing mitochondrial dysfunction. Such a mechanism of toxicity can be assumed taking into consideration that GLF neurotoxicity is induced by direct activation of NMDARs.
Uncertainties and Inconsistencies
Quantitative Understanding of the Linkage
Is it known how much change in the first event is needed to impact the second? Are there known modulators of the response-response relationships? Are there models or extrapolation approaches that help describe those relationships?
It was established that the dendritic calcium levels could underlie the differential vulnerability of C57BL/6 (resistant to kainite excitotoxicity) and C57BL/10 strains (vulnerable) mice to triggered neuronal degeneration induced by increased Ca2+ levels (Shuttleworth and Connor, 2001). A striking difference was found in dendrite calcium responses in hippocampus after kainate exposure of C57BL/6 (resistant to kainite excitotoxicity) and C57BL/10 strains (vulnerable). Ca2+ signals in distal dendrites were large in C57BL/10 neurons, and, if a threshold concentration of 1.5 uM was reached, a region of sustained high Ca2+ was established in the distal dendritic tree. This region then served as an initiation site for a degenerative cascade, producing high Ca2+ levels that slowly spread to involve the entire neuron and led to neuronal cell death. Dendritic Ca2+ signals in C57BL/6 neurons were much smaller and did not trigger these propagating secondary responses. Neurons from both strains had similar membrane properties and responded to kainate with intense action potential firing. Degenerative Ca2+ responses were seen in both strains if soma calcium level was above 1.5 uM serving as a threshold that if exceeded triggered excitotoxic neuronal cell death (Shuttleworth and Connor, 2001).
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
DomA toxicosis in California sea lions (CSLs, Zalophus californianus) is accompanied by increased expression of markers of oxidative stress such as malondialdehyde (MDA) and 3-nitrotyrosine (NT) in neurons (Madl et al., 2014).
In Atlantic salmon (Salmo salar), the cognition function has been investigated after exposure to sub-lethal doses of DomA (6 mg DA/kg bw). In addition, 14C-2-deoxyglucose has been injected i.m. to measure brain metabolic activity by autoradiography. The three brain regions investigated telencephalon, optic tectum and cerebellum have demonstrated a clear increase of metabolic activity in DomA exposed brains (Bakke and Horsberg, 2007).
Ananth C, Gopalakrishnakone P, Kaur C., Protective role of melatonin in domoic acid-induced neuronal damage in the hippocampus of adult rats. Hippocampus, 2003, 13: 375-87.
Attwell D, Laughlin SB, An energy budget for signaling in the grey matter of the brain. J. Cereb. Blood Flow Metab., 2001, 21: 1133–1145.
Bakke MJ, Horsberg TE., Effects of algal-produced neurotoxins on metabolic activity in telencephalon, optic tectum and cerebellum of Atlantic salmon (Salmo salar). Aquat Toxicol., 2007, 85: 96-103.
Choi IY, Lim JH, Kim C, Song HY, Ju C, Kim WK., 4-hydroxy-2(E)-Nonenal facilitates NMDA-Induced Neurotoxicity via Triggering Mitochondrial Permeability Transition Pore Opening and Mitochondrial Calcium Overload. Exp Neurobiol., 2013, 22 :200-207.
Giordano G, White CC, McConnachie LA, Fernandez C, Kavanagh TJ, Costa LG. Neurotoxicity of domoic Acid in cerebellar granule neurons in a genetic model of glutathione deficiency. Mol Pharmacol., 2006, 70 :2116-26.
Giordano G, White CC, Mohar I, Kavanagh TJ, Costa LG. Glutathione levels modulate domoic acid-induced apoptosis in mouse cerebellar granule cells. Toxicol Sci., 2007, 100: 433-444.
Gleichmann M, Mattson MP., Neuronal calcium homeostasis and dysregulation. Antioxid Redox Signal., 2011, 14 :1261-1273.
Lu J, Wu DM, Zheng YL, Hu B, Cheng W, Zhang ZF. Purple sweet potato color attenuates domoic acid-induced cognitive deficits by promoting estrogen receptor-α-mediated mitochondrial biogenesis signaling in mice. Free Radic Biol Med., 2012, 52: 646-59.
Lu J, Wu DM, Zheng YL, Hu B, Cheng W, Zhang ZF, Li MQ., Troxerutin counteracts domoic acid-induced memory deficits in mice by inhibiting CCAAT/enhancer binding protein β-mediated inflammatory response and oxidative stress. J Immunol., 2013, 190: 3466-3479.
Madl JE, Duncan CG, Stanhill JE, Tai PY, Spraker TR, Gulland FM., Oxidative stress and redistribution of glutamine synthetase in California sea lions (Zalophus californianus) with domoic acid toxicosis. J Comp Pathol., 2014, 150: 306-315.
Nicholls DG., Mitochondrial calcium function and dysfunction in the central nervous system. Biochim Biophys Acta., 2009, 1787: 1416-1424.
Peng TI, Jou MJ. Oxidative stress caused by mitochondrial calcium overload. Ann N Y Acad Sci., 2010, 201: 183-188.
Pivovarova NB, Nguyen HV, Winters CA, Brantner CA, Smith CL, Andrews SB., Excitotoxic calcium overload in a subpopulation of mitochondria triggers delayed death in hippocampal neurons. J Neurosci., 2004, 24: 5611-5622.
Rintoul GL, Filiano AJ, Brocard JB, Kress GJ, Reynolds IJ., Glutamate decreases mitochondrial size and movement in primary forebrain neurons. J Neurosci., 2003, 23: 7881-7888.
Shalbuyeva N, Brustovetsky T, Bolshakov A, Brustovetsky N. Calcium-dependent spontaneously reversible remodeling of brain mitochondria. J Biol Chem., 2006, 281: 37547-37558.
Shuttleworth CW, Connor JA. Strain-dependent differences in calcium signaling predict excitotoxicity in murine hippocampal neurons. J Neurosci., 2001, 15:21(12):4225-36.
Wu DM, Lu J, Zhang YQ, Zheng YL, Hu B, Cheng W, Zhang ZF, Li MQ., Ursolic acid improves domoic acid-induced cognitive deficits in mice. Toxicol Appl Pharmacol., 2013, 271: 127-36.