Mitochondrial Calcium Uniporter Inhibitor Research Articles

P32, a novel binding partner of Mcl-1, positively regulates mitochondrial Ca2+ uptake and apoptosis

Mcl-1 is a major anti-apoptotic Bcl-2 family protein. It is well known that Mcl-1 can interact with certain pro-apoptotic Bcl-2 family proteins in normal cells to neutralize their pro-apoptotic functions, thus prevent apoptosis. In addition, it was recently found that Mcl-1 can also inhibit mitochondrial calcium uptake. The detailed mechanism, however, is still not clear. Based on Yeast Two-Hybrid screening and co-immunoprecipitation, we identified a mitochondrial protein p32 (C1qbp) as a novel binding partner of Mcl-1. We found that p32 had a number of interesting properties: (1) p32 can positively regulate UV-induced apoptosis in HeLa cells. (2) Over-expressing p32 could significantly promote mitochondrial calcium uptake, while silencing p32 by siRNA suppressed it. (3) In p32 knockdown cells, Ruthenium Red treatment (an inhibitor of mitochondrial calcium uniporter) showed no further suppressive effect on mitochondrial calcium uptake. In addition, in Ruthenium Red treated cells, Mcl-1 also failed to suppress mitochondrial calcium uptake. Taken together, our findings suggest that p32 is part of the putative mitochondrial uniporter that facilitates mitochondrial calcium uptake. By binding to p32, Mcl-1 can interfere with the uniporter function, thus inhibit the mitochondrial Ca2+ uploading. This may provide a novel mechanism to explain the anti-apoptotic function of Mcl-1.

Role of mitochondrial calcium uniporter in regulating mitochondrial fission in the cerebral cortexes of living rats

The mitochondrial calcium uniporter (MCU) transports Ca2+ from the cytoplasm to the mitochondrial matrix and thus maintains Ca2+ homeostasis. Previous studies have reported that inhibition of MCU by ruthenium red (RR) protects the brain from ischemia/reperfusion (I/R) injury and that mitochondrial fission plays an important role in I/R injury. However, it is still not known whether MCU affects mitochondrial fission. In the present study, treatment with RR was found to decrease the concentration of free calcium in the mitochondria, calcineurin enzyme activity and dynamin-related protein 1 expression, and treatment with spermine was found to have the opposite effect in organisms subjected to occlusion of the middle cerebral artery lasting 2 h followed by 24 h reperfusion. These results indicate that MCU may be related to mitochondrial fission via modulating mitochondrial Ca2+ uptake and this relationship between MCU and mitochondrial fission may protect the brain from I/R injury.

MICU1 Motifs Define Mitochondrial Calcium Uniporter Binding and Activity

Resting mitochondrial matrix Ca(2+) is maintained through a mitochondrial calcium uptake 1 (MICU1)-established threshold inhibition of mitochondrial calcium uniporter (MCU) activity. It is not known how MICU1 interacts with MCU to establish this Ca(2+) threshold for mitochondrial Ca(2+) uptake and MCU activity. Here, we show that MICU1 localizes to the mitochondrial matrix side of the inner mitochondrial membrane and MICU1/MCU binding is determined by a MICU1 N-terminal polybasic domain and two interacting coiled-coil domains of MCU. Further investigation reveals that MICU1 forms homo-oligomers, and this oligomerization is independent of the polybasic region. However, the polybasic region confers MICU1 oligomeric binding to MCU and controls mitochondrial Ca(2+) current (IMCU). Moreover, MICU1 EF hands regulate MCU channel activity, but do not determine MCU binding. Loss of MICU1 promotes MCU activation leading to oxidative burden and a halt to cell migration. These studies establish a molecular mechanism for MICU1 control of MCU-mediated mitochondrial Ca(2+) accumulation, and dysregulation of this mechanism probably enhances vascular dysfunction.

Blockade of mitochondrial calcium uniporter prevents cardiac mitochondrial dysfunction caused by iron overload

Iron overload in the heart can lead to iron-overload cardiomyopathy and cardiac arrhythmia. In the past decades, growing evidence has suggested that cardiac mitochondrial dysfunction is associated with the development of cardiac dysfunction and lethal arrhythmias. Despite these facts, the effect of iron overload on cardiac mitochondrial function is still unclear. In this study, we determined the effects of iron overload on the cardiac mitochondrial function and the routes of cardiac mitochondrial iron uptake. We tested the hypothesis that iron overload can lead to cardiac mitochondrial dysfunction and that mitochondrial calcium uniporter (MCU) plays a major role for cardiac mitochondrial iron uptake under iron-overload condition. Cardiac mitochondrial function was assessed via the determination of mitochondrial swelling, mitochondrial reactive oxygen species (ROS) production and mitochondrial membrane potential changes. Isolated cardiac mitochondria from male Wistar rats were used in this study. To determine the routes for cardiac mitochondrial iron uptake, isolated mitochondria were exposed to MCU blocker (Ru360), mitochondrial permeability transition pore (mPTP) blocker (cyclosporin A) and an iron chelator (deferoxamine). We found that (i) iron overload caused cardiac mitochondrial dysfunction, indicated by increased ROS production, mitochondrial membrane depolarization and mitochondrial swelling; and (ii) only MCU blocker completely protected cardiac mitochondrial dysfunction caused by iron overload. These findings strongly suggest that MCU could be the major route for iron uptake into cardiac mitochondria. The inhibition of MCU could be the novel pharmacological intervention for preventing iron-overload cardiomyopathy.

Minocycline and doxycycline, but not other tetracycline-derived compounds, protect liver cells from chemical hypoxia and ischemia/reperfusion injury by inhibition of the mitochondrial calcium uniporter

Minocycline, a tetracycline-derived compound, mitigates damage caused by ischemia/reperfusion (I/R) injury. Here, 19 tetracycline-derived compounds were screened in comparison to minocycline for their ability to protect hepatocytes against damage from chemical hypoxia and I/R injury. Cultured rat hepatocytes were incubated with 50μM of each tetracycline-derived compound 20min prior to exposure to 500μM iodoacetic acid plus 1mM KCN (chemical hypoxia). In other experiments, hepatocytes were incubated in anoxic Krebs–Ringer–HEPES buffer at pH6.2 for 4h prior to reoxygenation at pH7.4 (simulated I/R). Tetracycline-derived compounds were added 20min prior to reperfusion. Ca2+ uptake was measured in isolated rat liver mitochondria incubated with Fluo-5N. Cell killing after 120min of chemical hypoxia measured by propidium iodide (PI) fluorometry was 87%, which decreased to 28% and 42% with minocycline and doxycycline, respectively. After I/R, cell killing at 120min decreased from 79% with vehicle to 43% and 49% with minocycline and doxycycline. No other tested compound decreased killing. Minocycline and doxycycline also inhibited mitochondrial Ca2+ uptake and suppressed the Ca2+-induced mitochondrial permeability transition (MPT), the penultimate cause of cell death in reperfusion injury. Ru360, a specific inhibitor of the mitochondrial calcium uniporter (MCU), also decreased cell killing after hypoxia and I/R and blocked mitochondrial Ca2+ uptake and the MPT. Other proposed mechanisms, including mitochondrial depolarization and matrix metalloprotease inhibition, could not account for cytoprotection. Taken together, these results indicate that minocycline and doxycycline are cytoprotective by way of inhibition of MCU.

The role of mitochondrial permeability transition pore in regulating the shedding of the platelet GPIbα ectodomain

Agonists induce platelet activation or apoptosis with concomitant shedding of the platelet receptor glycoprotein Ibα (GPIbα) ectodomain in a disintegrin and metalloproteinase 17 (ADAM17)-dependent mechanism. Mitochondrial permeability transition pore (MPTP) plays a pivotal role in platelet activation or apoptosis. However, its impact on ADAM17-mediated GPIbα ectodomain shedding remains unclear. Here, we aimed to test the hypothesis that MPTP regulates ADAM17-dependent GPIbα ectodomain shedding. We showed that calcium ionophore A23187- or thrombin plus collagen-induced GPIbα ectodomain shedding was partially inhibited by a MPTP inhibitor, and a MPTP potentiator promoted A23187-induced GPIbα cleavage. Furthermore, A23187-induced elevation of mitochondrial Ca2+ levels was blocked by the mitochondrial calcium uniporter (MCU) inhibitor Ru360 or treatment with the membrane-permeable Ca2+ chelator BAPTA-AM. We also demonstrated that an increase in mitochondrial Ca2+ levels triggered MPTP opening, as revealed by the examination of mitochondrial inner transmembrane potential depolarization. In addition, A23187 induced-mitochondrial reactive oxygen species (ROS) generation was blocked by the MPTP inhibitor or by treatment with the mitochondria-targeted ROS scavenger. Lastly, A23187-induced GPIbα shedding was partially blocked by inhibitors of either ROS or calpain, and was completely inhibited when platelets were exposed to both inhibitors. Therefore, these observations indicate that MPTP opening triggered mitochondrial ROS production, which plays an important role in regulating ADAM17-mediated shedding of the GPIbα ectodomain in A23187-treated platelets.

Abstract 230: Specific Mitochondrial Adaptation In Chronic Hypoxic Hearts Results In An Improved Intrinsic Energetic Homeostasis

Hypoxic states of the cardiovascular system are often associated with severe cardiac diseases. In spite of impairments in contractile activity, mitochondrial content, cell energy status and calcium transients, we recently demonstrated an improved energy homeostasis in chronic hypoxic hearts explained by a higher mitochondrial sensitivity to ATP/PCr changes, based on combined 31P-NMR spectroscopy and the analytical framework of metabolic control analysis. In the present study, we describe the role of calcium in co-variations in cellular energy status (PCr, ATP) and cardiac work (RPP), in perfused hearts from mice adapted for three weeks to a simulated altitude of 5500 m in a barochamber. Under basal conditions, lower cardiac work and PCr concentration confirmed energetic impairments in chronic hypoxic hearts. At high calcium concentration, cardiac work increased by 40% both in control and hypoxic hearts while a smaller PCr drop in hypoxic hearts, indicating an improved energy homeostasis and higher mitochondrial sensitivity to PCr changes. Stimulating calcium transients by adrenaline and thereby parallel activation of supply and demand components increased cardiac work twice as much only in control hearts (RPP +100%) without any significant PCr variation. The blunted effect of adrenaline in hypoxic hearts is likely the consequence of attenuated ß1-adrenergic receptors stimulation. Inhibition of mitochondrial calcium uniporter by ruthenium red which abolished the mitochondrial-mediated adrenergic regulation was not effective in chronic hearts in contrast of control. We conclude that although chronic hypoxic hearts failed to use calcium transients to optimize their inotropic response to a specific adrenergic stimulation, they present specific adaptation of the energetic homeostasis based on improved energy supply sensitivity. Interestingly this better regulation of the energetic system is not explained by a direct effect of calcium on mitochondria but rather by an optimized metabolic feedback.

The role of the mitochondrial calcium uniporter in cerebral ischemia/reperfusion injury in rats involves regulation of mitochondrial energy metabolism

The mitochondrial calcium uniporter (MCU) maintains intracellular Ca2+ homeostasis by transporting Ca2+ from the cell cytosol into the mitochondrial matrix and is important for shaping Ca2+ signals and the activation of programmed cell death. Inhibition of MCU by ruthenium red (RR) or Ru360 has previously been reported to protect against neuronal death. The aim of the present study was to analyze the mechanisms underlying the effects of MCU activity in a rat model of cerebral ischemia/reperfusion (I/R) injury. Adult male Wistar rats were divided into 4 groups; sham, I/R, I/R + RR and I/R + spermine (Sper) and were subjected to reversible middle cerebral artery occlusion for 2 h followed by 24 h of reperfusion. A bolus injection of RR administered 30 min prior to ischemia was found to significantly decrease the total infarct volume and reduce neuronal damage and cell apoptosis compared with ischemia/reperfusion values. However, treatment with Sper, an activator of the MCU, increased the injury induced by I/R. Analysis of energy metabolism revealed that I/R induced progressive inhibition of complexes I‑IV of the electron transport chain, decreased ATP production, dissipated the mitochondrial membrane potential and increased the generation of reactive oxygen species. Treatment with RR ameliorated the condition, while spermine had the opposite effect. In conclusion, blocking MCU was demonstrated to exert protective effects against I/R injury and this process may be mediated by the prevention of energy failure.

A possible pivotal role of mitochondrial free calcium in neurotoxicity mediated by N-methyl- d-aspartate receptors in cultured rat hippocampal neurons

We have previously shown that mitochondrial membrane potential disruption is involved in mechanisms underlying differential vulnerabilities to the excitotoxicity mediated by N-methyl- d-aspartate (NMDA) receptors between primary cultured neurons prepared from rat cortex and hippocampus. To further elucidate the role of mitochondria in the excitotoxicity after activation of NMDA receptors, neurons were loaded with the fluorescent dye calcein diffusible in the cytoplasm and organelles for determination of the activity of mitochondrial permeability transition pore (mPTP) responsible for the leakage of different mitochondrial molecules. The addition of CoCl 2 similarly quenched the intracellular fluorescence except mitochondria in both cultured neurons, while further addition of NMDA led to a leakage of the dye into the cytoplasm in hippocampal neurons only. An mPTP inhibitor prevented the NMDA-induced loss of viability in hippocampal neurons, while an activator of mPTP induced a similarly potent loss of viability in cortical and hippocampal neurons. Although NMDA was more effective in increasing rhodamine-2 fluorescence as a mitochondrial calcium indicator in hippocampal than cortical neurons, a mitochondrial calcium uniporter inhibitor significantly prevented the NMDA-induced loss of viability in hippocampal neurons. Expression of mRNA was significantly higher for the putative uniporter uncoupling protein-2 in hippocampal than cortical neurons. These results suggest that mitochondrial calcium uniporter would be at least in part responsible for the NMDA neurotoxicity through a mechanism relevant to promotion of mPTP orchestration in hippocampal neurons.

Role of mitochondrial calcium uniporter in cardioprotection induced by ischemic postconditioning in isolated rat heart

To investigate the role of mitochondrial calcium uniporter in cardioprotection elicited by ischemic postconditioning (Postcond). Male Sprague-Dawley rats were used for Langendorff isolated heart perfusion. The hearts subjected to global ischemia for 30 min followed by 120 min of reperfusion. Left ventricular developed pressure (LVDP), maximal rise/fall rate of left ventricular pressure (± dP/dtmax) were measured. The level of lactate dehydrogenase (LDH) in the coronary effluent was measured spectrophotometrically, the content of formazan of myocardium was also measured at the end of reperfusion. Compared to I/R group, Postcond had an significant increase in the mechanical function of the left ventricle, with LDH release reduced and the content of formazan increased. Spermine, the opener of mitochondrial calcium uniporter, deteriorated the mechanical function of left ventricle and decreased the formazan content, and increased LDH release. Ruthenium red, the inhibitor of mitochondrial calcium uniporter, increased the mechanical function of the left ventricle, decreased the LDH release, but the content of formazan was not increased. The inhibition of mitochondrial calcium uniporter is involved in the mechanisms of ischemic postconditioning.

Evidence for a Role for the Cytoskeleton in Communication Between the L-Type Calcium Channel and the Mitochondria in Isolated Cardiac Myocytes

Cytoskeletal proteins stabilize cell structure but also regulate subcellular distribution of mitochondria and cardiac L-type Ca2+ channel (LTCC) activity. We have previously demonstrated that mitochondrial function can be regulated by alterations in LTCC activity. This effect was attenuated when the cytoskeleton was disrupted with latrunculin A. To further explore this, we determined whether regulation of mitochondrial function by the LTCC is altered in a murine model of Duchenne Muscular Dystrophy(mdx). Mitochondrial membrane potential (Ψm) and metabolic activity was assessed after activation of the LTCC in cardiac myocytes isolated from C57BL/10ScSn-Dmdmdx/Arc (mdx) and C57BL/10ScSnArc (control) mice. Exposure of control myocytes to 10μM BayK(-) (LTCC agonist) caused a 11.4±1.7% increase in Ψm assessed as alterations in JC-1 compared to myocytes exposed to inactive BayK(+) (n=8, p<0.05). The response was attenuated when myocytes were exposed to LTCC antagonist nisoldipine (n=7). However BayK(-) did not induce any significant alteration in JC-1 signal in myocytes from mdx mice (n=6). In control myocytes BayK(-) caused a 105.4±7.4% increase in metabolic activity assessed using an MTT assay (n=8, p<0.05). The response was attenuated when myocytes were exposed to nisoldipine (n=8) or mitochondrial calcium uniporter inhibitor Ru360 (n=8) but unaltered when exposed to ryanodine receptor antagonist dantrolene (n=4). Exposure of mdx myocytes to BayK(-) did not induce any significant alteration in metabolic activity (n=8). These data confirm that alterations in LTCC activity can modulate mitochondrial function and that the cytoskeleton plays an important role in mediating this response. Since the LTCC is the initiator of contraction it has been proposed that a functional coupling between the LTCC and mitochondria may assist in meeting myocardial energy demand on a beat to beat basis.

Purification of the mitochondrial calcium uniporter from beef heart and characterization of its properties

A low-molecular-weight component (LMC) inducing selective transport of calcium across the bilayer lipid membrane has been isolated from mitochondria of the bovine heart by the method developed in our laboratory, which excludes the use of detergents and proteolytic enzymes. It was shown that, in the presence of 10 mM CaCl2, LMC forms conduction channels in the membrane multiples of 5 pS. The specific inhibitor of mitochondrial calcium uniporter, ruthenium red, closes Ca2(+)-induced channels formed in the membrane by LMC. In the absence of calcium or in the presence of potassium ions only, the component is incapable of forming channels of conduction. It was shown using nuclear magnetic resonance that LMC is a complex consisting of lipids, amino acids, and sugars with a molecular weight of 1-2 kDa.

Mechanism of cardioprotection induced by noninvasive limb ischemic preconditioning

To verify the inhibitory effect of mitochondrial calcium uniporter in remote preconditioning-induced cardioprotection. By occlusion and reperfusion of left anterior descending artery, the rat hearts were subjected to 30 min regional ischemia and 120 min reperfusion in vivo. Thus the ischemic reperfusion model was established. The rats were randomly assigned to undergo one of the following maneuvers: (1) remote preconditioning; (2) ruthenium red (an inhibitor of mitochondrial calcium uniporter); (3) spermine or SB202190 (an opener of mitochondrial calcium uniporter). Remote preconditioning was elicited by three cycles of 5 min of right femoral artery occlusion interspersed with 5 min of reperfusion. The mean arterial blood pressure, heart rate and lactate dehydrogenase released in plasma were measured during reperfusion but the infarct size was measured after reperfusion. In comparison with I/R group, remote preconditioning limited infarct size [(20.4 +/- 2.5)% vs (51.0 +/- 6.0)%] and lactate dehydrogenase release [(271 +/- 9) U/L vs (339 +/- 39)U/L] during reperfusion. On the contrary, spermine or SB202190 attenuated the reduction of infarct size and lactate dehydrogenase release induced by remote preconditioning. The group of spermine was [(40.8 +/- 9.2)% vs (20.4 +/- 2.5)%] and [(383 +/- 43) U/L vs (271 +/- 9) U/L] while the group of SB202190 was [(44.3 +/- 6.8)% vs (20.4 +/- 2.5)%] and [(356 +/- 26) U/L vs (271 +/- 9) U/L]. Inhibition of mitochondrial calcium uniporter opening is involved in the remote preconditioning-induced cardioprotection.

Tumor Necrosis Factor-α Potentiates Intraneuronal Ca2+ Signaling via Regulation of the Inositol 1,4,5-Trisphosphate Receptor

Inflammatory events have long been implicated in initiating and/or propagating the pathophysiology associated with a number of neurological diseases. In addition, defects in Ca2+-handling processes, which shape membrane potential, influence gene transcription, and affect neuronal spiking patterns, have also been implicated in disease progression and cognitive decline. The mechanisms underlying the purported interplay that exists between neuroinflammation and Ca2+ homeostasis have yet to be defined. Herein, we describe a novel neuron-intrinsic pathway in which the expression of the type-1 inositol 1,4,5-trisphosphate receptor is regulated by the potent pro-inflammatory cytokine tumor necrosis factor-alpha. Exposure of primary murine neurons to tumor necrosis factor-alpha resulted in significant enhancement of Ca2+ signals downstream of muscarinic and purinergic stimulation. An increase in type-1 inositol 1,4,5-trisphosphate receptor mRNA and protein steady-state levels following cytokine exposure positively correlated with this alteration in Ca2+ homeostasis. Modulation of Ca2+ responses arising from this receptor subtype and its downstream effectors may exact significant consequences on neuronal function and could underlie the compromise in neuronal activity observed in the setting of chronic neuroinflammation, such as that associated with Parkinson disease and Alzheimer disease.

Effect of protein kinase C?? inhibitor on Ca2+ homeostasis in diabetic sensory neurons

To elucidate the direct effect of selective protein kinase Cbeta inhibitor LY333531 on diabetic sensory neurons, we examined intracellular Ca(2+) concentration in isolated rat dorsal root ganglion neurons using the fluorescent Ca(2+) indicator fura-2. The duration of calcium transients induced by high (50 mM) extracellular K in small diabetic dorsal root ganglion neurons was significantly prolonged compared with that in control neurons. This prolonged intracellular Ca concentration elevation in diabetic neurons was normalized rapidly and reversibly by LY333531 in a dose-dependent manner, and the effect of LY333531 was completely abolished by pretreating the neurons with mitochondrial calcium uniporter inhibitor, Ruthenium 360. These results suggest that LY333531 has an ameliorating effect on calcium homeostasis of diabetic sensory neurons via mitochondrial calcium buffering.

Role of calcium-induced mitochondrial hydroperoxide in induction of apoptosis of RBL2H3 cells with eicosapentaenoic acid treatment

Eicosapentaenoic acid (EPA) was previously shown to induce caspase-independent apoptosis in rat basophilic leukemia cells (RBL2H3 cells) by translocation of apoptosis-inducing factor (AIF) [Free Radic Res (2005) 39, 225–235]. Here, we attempted to investigate the mechanism of EPA-induced apoptosis. A rapid and sustained increase in calcium was observed in mitochondria at 2 h after the addition of EPA prior to apoptosis. Coincidently, hydroperoxide was generated in the mitochondria after exposure to EPA. Production of mitochondrial hydroperoxide was significantly reduced by ruthenium red, an inhibitor of mitochondrial calcium uniporter, and BAPTA-AM, a cytoplasmic calcium chelator, indicating that generation of hydroperoxide is triggered by an accumulation of calcium in the mitochondria. The production of mitochondrial hydroperoxide was markedly attenuated by overexpression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in the mitochondria. Apoptosis was therefore, significantly prevented through inhibition of mitochondrial hydroperoxide generation with mitochondrial PHGPx, ruthenium red or BAPTA-AM. However, accumulation of calcium in the mitochondria was not prevented by mitochondrial PHGPx although apoptosis was blocked, indicating that elevated calcium does not directly induce apoptosis. Taken together, our results show that calcium-dependent hydroperoxide accumulation in the mitochondria is critical in EPA-induced apoptosis.

Critical role of mitochondrial damage in determining outcome of macrophage infection with Mycobacterium tuberculosis.

Human macrophages (Mphi) respond to Mycobacterium tuberculosis (Mtb) infection by undergoing apoptosis, a cornerstone of effective antimycobacterial host defense. Virulent mycobacteria override this reaction by inducing necrosis leading to uncontrolled Mtb replication. Accordingly, Mphi death induced by inoculation with Mtb had the characteristics of apoptosis and necrosis and correlated with moderate increase of mitochondrial permeability transition (MPT), mitochondrial cytochrome c release, and caspase-9 and -3 activation. We hypothesized that changes in intramitochondrial Ca(2+) concentration ([Ca(2+)](m)) determine whether Mphi undergo either apoptosis or necrosis. Therefore, we induced mechanism(s) leading to predominant apoptosis or necrosis by modulating [Ca(2+)](m) and examined their physiological consequences. Adding calcium ionophore A23187 to Mphi inoculated with Mtb further increased calcium flux into the cells which is thought to lead to increased [Ca(2+)](m), blocked necrosis, stabilized MPT, decreased mitochondrial cytochrome c release, lowered caspase activation, and accompanied effective antimycobacterial activity. In contrast, Mphi infected with Mtb in presence of the mitochondrial calcium uniporter inhibitor ruthenium red showed increased mitochondrial swelling and cytochrome c release and decreased MPT and antimycobacterial activity. Thus, in Mtb-infected Mphi, high levels of mitochondrial membrane integrity, low levels of caspase activation, and diminished mitochondrial cytochrome c release are hallmarks of apoptosis and effective antimycobacterial activity. In contrast, breakdown of mitochondrial membrane integrity and increased caspase activation are characteristic of necrosis and uncontrolled Mtb replication.

Cellular and mitochondrial changes in glutamate-induced HT4 neuronal cell death

Elevated levels of extracellular glutamate are neurotoxic. The cytotoxic property of extracellular glutamate is known to mediate two primary mechanisms, excitotoxicity and excitotoxicity-independent processes. The excitotoxicity-independent pathway was investigated in the current study in a mouse hippocampal-derived HT4 cell line. Exposure of HT4 cells to glutamate for 12 h induced loss of cell viability preceded by rapid loss of intracellular reduced glutathione followed by accumulation of intracellular reactive oxygen species, elevation of intracellular Ca 2+, progressive loss of mitochondrial membrane potential swelling and loss of mitochondrial outer membrane integrity. Glutamate-induced loss of DNA integrity has been detected. The antioxidants α-tocopherol and trolox, mitochondrial calcium uniporter inhibitor Ruthenium Red and protein synthesis inhibitor cycloheximide all showed protection against glutamate-induced toxicity. None of the protective agents except for α-tocopherol controlled the glutamate-induced reactive oxygen species build-up. However, these cell death regulators prevented the glutamate-induced mitochondrial damage and regulated glutamate-induced increase in intracellular Ca 2+. Carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone, a mitochondrial uncoupler, partially protected against glutamate-induced cell death and mitochondrial damage, while the mitochondrial ribosomal inhibitor chloramphenicol and extracellular Ca 2+ chelator ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid did not protect the cells against glutamate treatment. The results of this study demonstrated that mitochondrial dysfunction was a key event in the excitotoxicity-independent component of neuronal cell death. Reactive oxygen species accumulation and glutathione depletion were prominent in glutamate-treated cells; however, these events were not direct mediators of cell death.