Hierarchical recruitment by AMPA but not staurosporine of pro-apoptotic mitochondrial signaling in cultured cortical neurons: evidence for caspase-dependent/independent cross-talk |
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Authors: | Beart Philip M Lim Maria L R Chen Baohong Diwakarla Shanti Mercer Linda D Cheung Nam Sang Nagley Phillip |
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Institution: | Brain Injury and Repair Program, Howard Florey Institute, University of Melbourne, Parkville, Victoria, Australia; Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia; Department of Biochemistry and Molecular Biology, Monash University, Victoria, Australia; Department of Biochemistry, National University of Singapore, Singapore |
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Abstract: | 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. |
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Keywords: | (S)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate apoptosis Bax calpain caspase 3 mitochondrial membrane potential mitochondrial pro-apoptotic proteins |
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