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Hierarchical recruitment by AMPA but not staurosporine of pro‐apoptotic mitochondrial signaling in cultured cortical neurons: evidence for caspase‐dependent/independent cross‐talk

Excitotoxicity mediated via the (S)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) subtype of receptor for l‐glutamate contributes to various neuropathologies involving acute brain injury and chronic degenerative disorders. In this study, AMPA‐induced neuronal injury and staurosporine (STS)‐mediated apoptosis were compared in primary neuronal cultures of murine cerebral cortex by analyzing indices up‐ and downstream of mitochondrial activation. AMPA‐mediated apoptosis involved induction of Bax, loss of mitochondrial transmembrane potential (ΔΨm), early release of cytochrome c (cyt c), and more delayed release of second mitochondrial activator of caspases (SMAC), Omi, and apoptosis‐inducing factor (AIF) with early calpain and minor late activation of caspase 3. STS‐induced apoptosis was characterized by a number of differences, a more rapid time course, non‐involvement of ΔΨm, and relatively early recruitment of SMAC and caspase 3. The AMPA‐induced rise in intracellular calcium appeared insufficient to evoke ΔΨm as release of cyt c preceded mitochondrial depolarization, which was followed by the cytosolic translocation of SMAC, Omi, and AIF. Bax translocation preceded cyt c release for both stimuli inferring its involvement in apoptotic induction. Inclusion of the broad spectrum caspase inhibitor zVAD‐fmk reduced the AMPA‐induced release of cyt c, SMAC, and AIF, while only affecting the redistribution of Omi and AIF in the STS‐treated neurons. Only AIF release was affected by a calpain inhibitor (calpastatin) which exerted relatively minor effects on the progression of cellular injury. AMPA‐mediated release of apoptogenic proteins was more hierarchical relative to STS with its calpain activation and caspase‐dependent AIF redistribution arguing for a model with cross‐talk between caspase‐dependent/independent apoptosis.