Characterisation of calpain activation in response to excitotoxic events in primary neurons
Excitotoxicity resulting from excessive Ca²+ influx through glutamate receptors contributes to neuronal injury after stroke, trauma, and seizures. Increased cytosolic Ca²+ levels activate a family of calcium-dependent proteases with papain-like activity, the calpains. Here we investigated the role of calpain activation during NMDA-induced excitotoxic injury in embryonic (E16-E18) murine cortical neurons that underwent either: (i) excitotoxic necrosis, characterized by immediate deregulation of Ca²+ homeostasis, a persistent depolarization of mitochondrial membrane potential (Ay/m), and insensitivity to bax-gene deletion; (ii) excitotoxic apoptosis, characterized by recovery of NMDA-induced cytosolic Ca²+ increases, sensitivity to bax gene deletion, and delayed Ai//m depolarization and Ca²+ deregulation; or (iii) that were tolerant to excitotoxic injury. Interestingly, treatment with the calpain inhibitor calpeptin, overexpression of the endogenous calpain inhibitor calpastatin, or gene silencing of calpain protected neurons against excitotoxic apoptosis, but did not influence excitotoxic nccrosis. Calpeptin failed to exert a protective effect in oax-deficient neurons, but protected bid- and e/'m-deficient neurons similarly to wild-type cells. To identify when calpains became activated during excitotoxic apoptosis, we monitored calpain activation dynamics by time-lapse fluorescence microscopy, using a calpain-sensitive Forster resonance energy transfer probe.
We observed a delayed calpain activation that occurred downstream of mitochondrial engagement and directly preceded neuronal death. In contrast, we could not detect significant calpain activity during excitotoxic necrosis or in neurons that were tolerant to excitotoxic injuiy. Oxygen/glucose deprivationinduced injury in organotypic hippocampal slice cultures confirmed that calpains were specifically activated during .ax-dependent apoptosis and in this setting function as downstream cell death executioners. Lastly, box gene deletion prevented NMDA-induced alterations to Ay/m, hyperpolarisation and depolarisation were notably diminished, and affected neuronal calcium handling, resulting in increased neuronal survival.