Transient mitochondrial permeability transition mediates excitotoxicity in glutamate-sensitive NSC34D motor neuron-like cells

Abstract

Excitotoxicity plays a critical role in neurodegenerative disease. Cytosolic calcium overload and mitochondrial dysfunction are among the major mediators of high level glutamate-induced neuron death. Here, we show that the transient opening of mitochondrial permeability transition pore (tMPT) bridges cytosolic calcium signaling and mitochondrial dysfunction and mediates glutamate-induced neuron death. Incubation of the differentiated motor neuron-like NSC34D cells with glutamate (1mM) acutely induces cytosolic calcium transient (30% increase). Glutamate also stimulates tMPT opening, as reflected by a 2-fold increase in the frequency of superoxide flash, a bursting superoxide production event in individual mitochondria coupled to tMPT opening. The glutamate-induced tMPT opening is attenuated by suppressing cytosolic calcium influx and abolished by inhibiting mitochondrial calcium uniporter (MCU) with Ru360 (100 μM) or MCU shRNA. Further, increased cytosolic calcium is sufficient to induce tMPT in a mitochondrial calcium dependent manner. Finally, chronic glutamate incubation (24h) persistently elevates the probability of tMPT opening, promotes oxidative stress and induces neuron death. Attenuating tMPT activity or inhibiting MCU protects NSC34D cells from glutamate-induced cell death. These results indicate that high level glutamate-induced neuron toxicity is mediated by tMPT, which connects increased cytosolic calcium signal to mitochondrial dysfunction.

Keywords: Excitotoxicity; Glutamate; Mitochondrial calcium uniporter; Neuron death; Superoxide flashes; Transient mitochondrial permeability transition.

Fig. 1

Differentiated NSC34_D motor neuron cells responded to glutamate. A–C, Differentiation of the motor neuron cell line NSC34 cells to the differentiated NSC34_D cells, which exhibited long projections extended from soma (A), expressed glutamate receptor 2 (GluR2, B), and showed elevated cytosolic Ca²⁺ in response to glutamate (1 mM, arrow) (C). Scale bar in A, 50 µm. D, Glutamate increased cytosolic Ca²⁺ in NSC34_D cells, which was attenuated by pre-incubation (added 30 min before glutamate) of glutamate receptor inhibitors, MK801 (10 µM) or NBQX (1 µM). E, Inhibiting Ca²⁺ influx by removing extracellular Ca²⁺ attenuated glutamate-induced cytosolic Ca²⁺ increase. Arrows indicate the addition of glutamate (1 mM). Images in A and traces in C–E are representative of at least 10 cells/images from 3 independent experiments.

Fig. 2

Glutamate stimulated superoxide flash activity in motor neurons. A, Representative confocal images showed NSC34_D cells expressing mitochondrial targeted superoxide indicator, mt-cpYFP, in soma and neurites (excitation by 405 and 488 nm laser, Upper panel). The enlarged images showed mitochondrial expression pattern of mt-cpYFP in a neuron (Lower panel). Scale bars = 20 µm. B, Time lapse images of a small area of the neuron highlighted by red box in A at 405 or 488 nm excitation. The pseudo-colors indicate relative fluorescence intensity (F/F₀). A single mitochondrial superoxide flash event was detected at 488 nm excitation which peaked at 6–9 s, as shown by the dramatically increased fluorescence. Scale bar = 2 µm. C, 3D surface plot of the area shown in B and at the peak of the single mitochondrial superoxide flash. The same color code applies to both B and C. D, Representative traces of superoxide flashes in NSC34_D cells before and after glutamate (1 mM, 10 min) treatment. E, Summarized data showing flash frequency in NSC34 and NSC34_D before and after glutamate treatment (1 mM, 10 min). N = 6–8 cells. *, p < 0.05 by unpaired t test. Vertical bars are SEM. F-H, Flash amplitude (ΔF/F₀, F), time to peak (Tpk, G), and time to 50% decay (T₅₀, H) in NSC34_D cells before and after glutamate treatment. N = 65–117 flashes from 10 cells in each group. *, p < 0.05 by unpaired t test. Vertical bars are SEM.

Fig. 3

Transient openings of permeability transition pore (tMPT) underlay basal and glutamate-induced superoxide flash activity in motor neurons. A, Representative images of a NSC34_D cell expressing mt-cpYFP and loaded with mitochondrial membrane potential indicator, TMRM (20 nM). Scale bar = 20 µm. B, Traces showing a superoxide flash accompanied by mitochondrial membrane depolarization after glutamate (1 mM) treatment. C, Adenovirus-mediated human SOD2 expression in NSC34_D inhibited basal and glutamate-induced flash activity. N = 6–22 cells. *, p < 0.05 versus Control and #, p < 0.01 versus without Ad-SOD2 by ANOVA and unpaired t test. Vertical bars are SEM. D, tMPT inhibitor, cyclosporin A (1 µM), suppressed basal and glutamate-induced superoxide flash activity. N = 7–14 cells. *, p < 0.05 by ANOVA and unpaired t test. Vertical bars are SEM. E, tMPT activator, atractyloside (10 µM) stimulated flash activity in NSC34_D cells. N = 8 cells. *, p < 0.05 by unpaired t test. Vertical bar is SEM.

Fig. 4

Glutamate-induced superoxide flash activity required mitochondrial Ca²⁺ uptake during cytosolic Ca²⁺ increase. A, Glutamate receptor inhibitors, MK801 (10 µM) and NBQX (1 µM), blocked glutamate (1mM for 10 min)-induced superoxide flash activity. N = 11–12 cells. Vertical bars are SEM. The dashed line indicates the level of glutamate-induced flash frequency (Mean value of the NSC34_D + glutamate group in Figure 2E), which also applies for B, C and E. B-C, Glutamate-induced superoxide flash activity was attenuated by removal of extracellular Ca²⁺ (B), and was abolished by inhibiting mitochondrial Ca²⁺ uniporter (MCU) by Ru360 (100 µM, C). N = 10–14 cells. Vertical bars are SEM. Note while removing extracellular Ca²⁺, no Ca²⁺ chelator was added, and residual Ca²⁺ may exist in the solution, which could be responsible for the slight but insignificant increase of flash events by glutamate in B. D, Lentivirus-mediated expression of MCU shRNA decreased protein levels of MCU and abolished glutamateinduced flash activity in NSC34_D cells. N = 14 cells from 3 independent experiments. Vertical bars are SEM. E, Lentivirus-mediated expression of a control scrambled shRNA sequence (SC shRNA) has no effect on MCU levels, and basal or 1 mM glutamate-induced flash frequency in NSC34_D cells. N = 12–16 cells from 3 independent experiments. *, p < 0.05 versus Basal by unpaired t test. Vertical bars are SEM.

Fig. 5

Cytosolic Ca²⁺ leads to mitochondrial Ca²⁺ uptake and increased superoxide flashes in motor neurons. A, Representative image showing a NSC34_D cell loaded with Ca²⁺ indicator, Fluo-4, in cytosol (soma and neurites). Scale bar = 50 µm. B, Trace showing Ca²⁺ ionophore, A23187 (1 µM, 10 min) increased cytosolic Ca²⁺ in NSC34_D cells. C-D, A23187 stimulated superoxide flash activity (C), which was blocked by mitochondrial Ca²⁺ uniporter inhibitor, Ru360 (100 µM, D). N = 8–10 cells. *, p < 0.05 by t test. Vertical bars are SEM.

Fig. 6

Chronic glutamate treatment continuously stimulated superoxide flash activity and induced oxidative mitochondrial dysfunction and neuron death. A, Superoxide flash activity was increased by glutamate (1 mM) treatment for 24 hr and decreased after 48 hr treatment. N = 7–20 cells. *, p < 0.05 by ANOVA and unpaired t test. Vertical bars are SEM. B, Glutamate (1 mM) treatment for 24 hr increased mitochondrial and cytosolic oxidative stress as shown by increased MitoSOX and DCF signals, respectively. N = 24–30 cells. *, p < 0.05 by unpaired t test. Vertical bars are SEM. C, Mitochondrial membrane depolarization after 24 hr glutamate (1 mM) treatment as shown by decreased JC-1 red/green ratio. N = 39–42 cells. *, p < 0.05 by unpaired t test. Vertical bars are SEM. D-F, Chronic and high level glutamate treatment (1 mM, 24–48 hr) induced NSC34_D cell death (D), which was blocked by glutamate receptor antagonists, tMPT blockade (E) and MCU shRNA (F). N = 13–26 images from 3 independent measurements. Each image contains 50–100 cells. *, p < 0.05 by ANOVA and unpaired t test. Vertical bars are SEM.

Fig. 7

Schematic model showing the mitochondrial Ca²⁺-tMPT-superoxide flash pathway that underlies the effects of glutamate in motor neurons. Glutamate elevates cytosolic Ca²⁺, which promotes mitochondrial Ca²⁺ uptake to trigger tMPT opening and the bursting superoxide flashes. Under physiological conditions, this pathway is likely transiently activated. Under pathological conditions, chronic or high level of glutamate may significantly activate this pathway and lead to oxidative stress, mitochondrial dysfunction and neuron death.