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)-alpha-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 (deltapsi(m)), 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 deltapsi(m), and relatively early recruitment of SMAC and caspase 3. The AMPA-induced rise in intracellular calcium appeared insufficient to evoke feltapsi(m) 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.
Beart
Philip M
PM
Brain Injury and Repair Program, Howard Florey Institute, University of Melbourne, Parkville, Victoria, Australia. p.beart@hfi.unimelb.edu.au
Lim
Maria L R
ML
Chen
Baohong
B
Diwakarla
Shanti
S
Mercer
Linda D
LD
Cheung
Nam Sang
NS
Nagley
Phillip
P
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins / Q000187:drug effects / Q000378:metabolism; D002154:Calpain / Q000378:metabolism; D020169:Caspases / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009498:Neurotoxins / Q000494:pharmacology; D020239:Receptor Cross-Talk / Q000187:drug effects / Q000502:physiology; D018091:Receptors, AMPA / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D019311:Staurosporine / Q000494:pharmacology; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology
D051017:Apoptosis Regulatory Proteins; D004791:Enzyme Inhibitors; D018690:Excitatory Amino Acid Agonists; D009498:Neurotoxins; D018091:Receptors, AMPA; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D045304:Cytochromes c; D002154:Calpain; D020169:Caspases; D019311:Staurosporine