Pharmacological Sequestration of Mitochondrial Calcium Uptake Protects Neurons Against Glutamate Excitotoxicity

Abstract

Neuronal excitotoxicity which is induced by exposure to excessive extracellular glutamate is shown to be involved in neuronal cell death in acute brain injury and a number of neurological diseases. High concentration of glutamate induces calcium deregulation which results in mitochondrial calcium overload and mitochondrial depolarization that triggers the mechanism of cell death. Inhibition of mitochondrial calcium uptake could be potentially neuroprotective but complete inhibition of mitochondrial calcium uniporter could result in the loss of some physiological processes linked to Ca²⁺ in mitochondria. Here, we found that a novel compound, TG-2112x, can inhibit only the lower concentrations mitochondrial calcium uptake (induced by 100 nM-5 μM) but not the uptake induced by higher concentrations of calcium (10 μM and higher). This effect was not associated with changes in mitochondrial membrane potential and cellular respiration. However, a pre-treatment of neurons with TG-2112x protected the neurons against calcium overload upon application of toxic concentrations of glutamate. Thus, sequestration of mitochondrial calcium uptake protected the neurons against glutamate-induced mitochondrial depolarization and cell death. In our hands, TG-2112x was also protective against ionomycin-induced cell death. Hence, low rate mitochondrial calcium uptake plays an underestimated role in mitochondrial function, and its inhibition could protect neurons against calcium overload and cell death in glutamate excitotoxicity.

Keywords: Calcium; Excitotoxicity; Glutamate; Mitochondria; Neuron.

Fig. 1

Structure of the compound TG-2112x

Fig. 2

Effect of TG-2112x on [Ca²⁺]_c and mitochondrial membrane potential during 100 μM glutamate exposure. Simultaneous measurements of the changes in [Ca²⁺]_c (Fura-ff ratio) and Δψ_m (relative Rh 123 fluorescence) were made from single neurons. An increase in the Rh123 fluorescence reflects mitochondrial depolarization which corresponds to the effect of mitochondrial uncoupler FCCP (1 μM) which was applied at the end of this and subsequent experiments to reflect complete dissipation of Δψ_m. a, b. illustration of the differences in the dynamics of [Ca²⁺]_c and Δψ_m in control (a) and in response to pre-incubation of the cells with 0.5 μM TG-2112x (b). c Summaries of the effects of the different concentration of TG-2112x on glutamate-induced mitochondrial depolarization (n = 88 neurons for control and 67, 59, and 89 cells for 0.5, 1, and 5 μM TG-2112x). **p < 0.001

Fig. 3

Effect of TG-2112 on [Ca²⁺]_c changes in neurons under application of physiological concentration of glutamate. Pre-incubation of cells with 0.5 μM (b) or 1 μM (c) TG-2112x increased the effect of 5 μM glutamate on [Ca²⁺]_c neurons compare to control (a). c Summary of the effects of TG-2112x on glutamate-induced calcium signal shown as a difference in Fura-2 ratio. (n = 67 cells- in control; n = 56 cells (0.5 μM TG-2112x); n = 76 cells (1 μM) **p < 0.001

Fig. 4

TG-2112x had no effect of the glutamate-induced mitochondrial calcium overload. Glutamate induced delayed calcium deregulation in control (a, aii) and in TG-2112x (0.5 μM; b, bii)-treated neurons measured with low affinity calcium indicator Rhod-5n. Effect 100 μM on mitochondrial calcium is impossible to detect in cell body due to DCD, but the effect of TG-2112x on glutamate-induced calcium uptake is easily detectable in processi (aii, bii). Traces represent individual cells (measured from cytosolic area ai) or mitochondria (bi). c Summary of the cytosolic responses to glutamate in control (n = 122 neurons) and treated with TG-2112x (0.5 μM; n = 99 neurons)

Fig. 5

TG-2112x protects neurons against glutamate-induced mitochondrial calcium overload. TG-2112x (0.5 μM) blocks the calcium rise in majority of the mitochondria in the neuronal processi (b, bii) compare to control neurons (a, aii). Note that rise in rhod-5n signal in TG-2112x-treated neurons has recovered after an initial peak compared to the control neurons. The traces represent individual mitochondria from the processi. c Summary of the mitochondrial responses to glutamate in control (n = 122 neurons) and treated with TG-2112x (0.5 μM; n = 99). **p < 0.001. d (representative traces), dii (representative images)—effect of 5 μM glutamate in control (n = 26 neurons), TG-2112x-treated cells (n = 35) and neurons treated with 10 μM Ru360 (n = 21 neuron) measured with genetically-encoded indicator for mitochondrial calcium MitoGCaMP6

Fig. 6

Effect of TG-2112x on the mitochondrial calcium uptake. TG-2112x inhibits calcium-induced swelling of isolated rat brain mitochondria (a). TG-2112x influence on the calcium retention capacity in isolated rat brain mitochondria with different respiratory substrates (b 5 mM glutamate/malate, c 5 mM succinate with complex I inhibitor 1 μM rotenone). Application of 100 nM-5 μM calcium to mitochondria in permeabilized neurons and astrocytes induced rise in [Ca²⁺]_m in control (d, n = 5 experiments), but not in TG-2112x (0.5 μM; n = 4 experiments)-treated mitochondria (e)

Fig. 7

TG-2112x does not induce mitochondrial dysfunction. TG-2112x induced mild hyperpolarization of mitochondria (resulted in a decrease of Rh123 fluorescence; a; n = 4 experiments), decrease of mitochondrial NADH autofluorescence (b; n = 4 experiments), and increase in FAD++ fluorescence (c; n = 5 experiments) shown as representative traces from the single cells. TG-2112x did not influence the basal oxygen consumption rate (OCR), non-mitochondrial respiration of mixed cultures of cortical neurones, and glial cells (d lines represents the mean value traces of different experiments). OM oligomycin (3 μM), FCCP (3 μM), AA/Rot rotenone (1 μM) with antimycin (1 μM), but a tendency to an increased respiratory capacity in the presence of the mitochondrial uncoupler FCCP was observed (e; n = 5 of each experiments)

Fig. 8

TG-2112x protects neurons against calcium-induced cell death. Pre-incubation of the cortical neurons with 0.5 μM TG2112x protects cells against cell death, induced by 100 μM glutamate (a). TG-2112x dose-dependently protects cerebellar granular cells against ionomycin-induced neuronal death (b), n = 4 of each experiments; **p < 0.001; ***p < 0.0001