CGP37157, an inhibitor of the mitochondrial Na+/Ca2+ exchanger, protects neurons from excitotoxicity by blocking voltage-gated Ca2+ channels

Abstract

Inhibition of the mitochondrial Na+/Ca2+ exchanger (NCLX) by CGP37157 is protective in models of neuronal injury that involve disruption of intracellular Ca2+ homeostasis. However, the Ca2+ signaling pathways and stores underlying neuroprotection by that inhibitor are not well defined. In the present study, we analyzed how intracellular Ca2+ levels are modulated by CGP37157 (10 μM) during NMDA insults in primary cultures of rat cortical neurons. We initially assessed the presence of NCLX in mitochondria of cultured neurons by immunolabeling, and subsequently, we analyzed the effects of CGP37157 on neuronal Ca2+ homeostasis using cameleon-based mitochondrial Ca2+ and cytosolic Ca2+ ([Ca2+]i) live imaging. We observed that NCLX-driven mitochondrial Ca2+ exchange occurs in cortical neurons under basal conditions as CGP37157 induced a decrease in [Ca2]i concomitant with a Ca2+ accumulation inside the mitochondria. In turn, CGP37157 also inhibited mitochondrial Ca2+ efflux after the stimulation of acetylcholine receptors. In contrast, CGP37157 strongly prevented depolarization-induced [Ca2+]i increase by blocking voltage-gated Ca2+ channels (VGCCs), whereas it did not induce depletion of ER Ca2+ stores. Moreover, mitochondrial Ca2+ overload was reduced as a consequence of diminished Ca2+ entry through VGCCs. The decrease in cytosolic and mitochondrial Ca2+ overload by CGP37157 resulted in a reduction of excitotoxic mitochondrial damage, characterized here by a reduction in mitochondrial membrane depolarization, oxidative stress and calpain activation. In summary, our results provide evidence that during excitotoxicity CGP37157 modulates cytosolic and mitochondrial Ca2+ dynamics that leads to attenuation of NMDA-induced mitochondrial dysfunction and neuronal cell death by blocking VGCCs.

Inorganic polyphosphate (polyP) is required for sustained free mitochondrial calcium elevation, following stimulated calcium uptake.

“…Another possibility that might explain the differences observed in the mitochondrial free calcium signal is the presence of increased calcium efflux in the MitoPPX cells. To test this, we measured the calcium response to 100µM ATP in the presence of 10µM CGP-37157, a well-known inhibitor of the mitochondrial sodium-calcium exchanger, (NCLX) (Ruiz et al, 2014). Importantly, NCLX is the primary pathway involved on mitochondrial calcium efflux (Palty et al, 2010).…”

Standing of giants shoulders the story of the mitochondrial Na+Ca2+ exchanger

“…Similarly, other studies have shown that CGP-37157 may also modulate the activity of other transporters, among them the Ca 2þ pump and the ryanodine receptor [19]. Recent studies further suggest that the neuronal rescue effect attributed to prevention of reversal of the mitochondrial Na þ /Ca 2þ exchanger is in fact related to LTCC block [20]. Thus, a selective molecular tool was required for studying the role of the exchanger.…”

Life after the birth of the mitochondrial Na+/Ca2+ exchanger, NCLX

“…While CGP-37157 effectively reduced the mitochondrial Ca 2+ efflux, it also reduced the cytosolic Ca 2+ rise and neuronal cell death. The latter effect could be fully reproduced by nifedipine, a classical L-Type Ca 2+ channel blocker that does not modulate NCLX activity [32]. This study, therefore, warrants reconsideration of the role of the exchanger in neuronal survival by molecular tools aimed at controlling NCLX expression or by an NCLX knockdown mouse model.…”