Increased, Intracellular Calcium overload leads to Mitochondrial dysfunction

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).

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.