Calpain activation and Na⁺/Ca²⁺ exchanger degradation occur downstream of calcium deregulation in hippocampal neurons exposed to excitotoxic glutamate

Abstract

Delayed calcium deregulation (DCD) plays an essential role in glutamate excitotoxicity, a major detrimental factor in stroke, traumatic brain injury, and various neurodegenerations. In the present study, we examined the role of calpain activation and Na+/Ca2+ exchanger (NCX) degradation in DCD and excitotoxic cell death in cultured hippocampal neurons. Exposure of neurons to glutamate caused DCD accompanied by secondary mitochondrial depolarization. Activation of calpain was evidenced by detecting NCX isoform 3 (NCX3) degradation products. Degradation of NCX isoform 1 (NCX1) was below the detection limit of western blotting. Degradation of NCX3 was detected only after an hour of incubation with glutamate, while DCD occurred on average within 15 minutes after glutamate application. Calpeptin, an inhibitor of calpain, significantly attenuated NCX3 degradation but failed to inhibit DCD and excitotoxic neuronal death. Calpain inhibitors I, III, and VI also failed to influence DCD and glutamate-induced neuronal death. On the other hand, MK801, an inhibitor of the NMDA-subtype of glutamate receptors, added shortly after the initial glutamate-induced jump in cytosolic Ca2+, completely prevented DCD and activation of calpain and strongly protected neurons against excitotoxicity. Taken together, our results suggest that, in glutamate-treated hippocampal neurons, the initial increase in cytosolic Ca2+ that precedes DCD is insufficient for sustained calpain activation, which most likely occurs downstream of DCD.

Delayed calcium dysregulation in neurons requires both the NMDA receptor and the reverse Na+/Ca2+ exchanger

“…In our previous study (Brustovetsky et al, 2010), MK801, a noncompetitive NMDAR inhibitor, prevented glutamate-induced DCD, consistent with the results from others (Stanika et al, 2009). These data suggested that NMDAR plays a key role in DCD.…”

“…In our previous study, we found that the NMDAR inhibitor, MK801, applied to neurons shortly after glutamate, completely prevented DCD (Brustovetsky et al, 2010). This finding supports the major role of NMDAR in DCD and apparently rejects the NCX rev hypothesis.…”

Calpains and Delayed Calcium Deregulation in Excitotoxicity

“…dye and observed that calpain activation only occurs after DCD and mitochondrial depolarization take place, suggesting that calpain activation is not required for DCD in the model used in this study. Brustovetsky et al [24] reached a similar conclusion by observing NCX3 cleavage in hippocampal neurons only 45 min after the onset of DCD, suggesting that NCX3 cleavage is not required for DCD; moreover, several calpain inhibitors used in this study, although preventing NCX proteolysis, failed to prevent DCD, supporting the fact that NCX proteolysis by calpain is independent of DCD.…”

Suppression of Immunodeficiency Virus-Associated Neural Damage by the p75 Neurotrophin Receptor Ligand, LM11A-31, in an In Vitro Feline Model

“…Our previous studies suggested a dysfunction of the sodium-calcium exchanger (NCX) may contribute to the calcium dysregulation(Bragg et al, 2002; Meeker et al, 2005). The role of forward and reverse calcium exchange in different settings is still controversial (Bano et al, 2005; Brustovetsky et al, 2010) and the relationship between the calcium dysregulation and structural pathology is unclear. Various hypotheses have implicated dysfunctional mitochondria (Greenwood et al, 2007), loss of actin structure(Zeng et al, 2007) or disruption of neurofilaments (Kim et al, 2010).…”