TNF-α stimulates caspase-3 activation and apoptotic cell death in primary septo-hippocampal cultures
✍ Scribed by Xiurong Zhao; Brian Bausano; Brian R. Pike; Jennifer K. Newcomb-Fernandez; Kevin K.W. Wang; Esther Shohami; N.C. Ringger; S.M. DeFord; Douglas K. Anderson; Ronald L. Hayes
- Publisher
- John Wiley and Sons
- Year
- 2001
- Tongue
- English
- Weight
- 902 KB
- Volume
- 64
- Category
- Article
- ISSN
- 0360-4012
- DOI
- 10.1002/jnr.1059
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✦ Synopsis
Abstract
Primary septo‐hippocampal cell cultures were incubated in varying concentrations of tumor necrosis factor (TNF‐α; 0.3–500 ng/ml) to examine proteolysis of the cytoskeletal protein α‐spectrin (240 kDa) to a signature 145 kDa fragment by calpain and to the apoptotic‐linked 120‐kDa fragment by caspase‐3. The effects of TNF‐α incubation on morphology and cell viability were assayed by fluorescein diacetate‐propidium iodide (FDA‐PI) staining, assays of lactate dehydrogenase (LDH) release, nuclear chromatin alterations (Hoechst 33258), and internucleosomal DNA fragmentation. Incubation with varying concentrations of TNF‐α produced rapid increases in LDH release and nuclear PI uptake that were sustained over 48 hr. Incubation with 30 ng/ml TNF‐α yielded maximal, 3‐fold, increase in LDH release and was associated with caspase‐specific 120‐kDa fragment but not calpain‐specific 145‐kDa fragment as early as 3.5 hr after injury. Incubation with the pan‐caspase inhibitor, carbobenzosy‐ Asp‐CH~2~‐OC (O)‐2‐6‐dichlorobenzene (Z‐D‐DCB, 50‐140 μM) significantly reduced LDH release produced by TNF‐α. Apoptotic‐associated oligonucleosomal‐sized DNA fragmentation on agarose gels was detected from 6 to 72 hr after exposure to TNF‐α. Histochemical changes included chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies. Results of this study suggest TNF‐α may induce caspase‐3 activation but not calpain activation in septo‐hippocampal cultures and that this activation of caspase‐3 at least partially contributes to TNF‐α‐induced apoptosis. J. Neurosci. Res. 64:121–131, 2001. © 2001 Wiley‐Liss, Inc.