TNF-alpha stimulates caspase-3 activation and apoptotic cell death in primary septo-hippocampal cultures

Primary septo-hippocampal cell cultures were incubated in varying concentra tions of tumor necrosis factor (TNF-alpha; 0.3-500 ng/ml) to examine proteo lysis of the cytoskeletal protein alpha -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-alpha incubation on morphology and cell viab ility were assayed by fluorescein diacetate-propidium iodide (FDA-PI) stain ing, assays of lactate dehydrogenase (LDH) release, nuclear chromatin alter ations (Hoechst 33258), and internucleosomal DNA fragmentation. Incubation with varying concentrations of TNF-alpha produced vapid increases in LDH re lease and nuclear PI uptake that were sustained over 48 hr. Incubation with 30 ng/ml TNF-alpha yielded maximal, 3-fold, increase in LDH release and wa s associated with caspase-specific 120-kDa fragment but not calpain-specifi c 145-kDa fragment as early as 3.5 hr after injury. Incubation with the pan -caspase inhibitor, carbobenzosy-Asp-CH2-OC (O)-2-6-dichlorobenzene (Z-D-DC B, 50-140 muM) significantly reduced LDH release produced by TNF-alpha. Apo ptotic-associated oligonucleosomal-sized DNA fragmentation on agarose gels was detected from 6 to 72 hr after exposure to TNF-alpha. Histochemical cha nges included chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies. Results of this study suggest TNf-alpha may induce cas pase-3 activation but not calpain activation in septo-hippocampal cultures and that this activation of caspase-3 at least partially contributes to TNF -alpha -induced apoptosis. J. Neurosci. Res. 64,121-131, 2001. (C) 2001 Wil ey-Liss, Inc.