Journal of Neurochemistry, 2001, 76, 520±531 Neuronal death is an active, caspase-dependent process after moderate but not severe DNA damage Glenn T. Gobbel* and Pak H. Chan² *Department of Neurological Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA ²Departments of Neurosurgery, Neurology, and Neurological Sciences , Program in Neurosciences, Stanford University, Palo Alto, California, USA Abstract Mild insults to neurons caused by ischemia or glutamate induce apoptosis, whereas severe insults induce non apop- totic death, such as necrosis. The molecular targets that are damaged by these insults and ultimately induce cell death are not fully established. To determine if DNA damage can induce apoptotic or non apoptotic death depending on the severity, neurons were treated with up to 128 Gy of ionizing radiation. Such treatment induced a dose-related increase in DNA single-strand breaks but no immediate membrane disruption or lipid peroxidation. Following moderate doses of < 32 Gy, neuronal death had many characteristics of apoptosis includ- ing nuclear fragmentation and DNA laddering. Nuclear fragmentation and membrane breakdown after moderate DNA damage could be blocked by inhibition of active protein synthesis with cycloheximide and by inhibition of caspases. In contrast, cell death after doses of . 32 Gy was not blocked by cycloheximide or caspase inhibitors, and membrane break- down occurred relatively early in the cell death process. These data suggest that cell death after high dose irradiation and severe DNA damage can occur by non apoptotic mechanisms and that blocking apoptotic pathways may not prevent death after severe damage. Keywords: apoptosis, caspase, DNA damage, ionizing radiation, lipid peroxidation, neuron. J. Neurochem. (2001) 76, 520±531. Insults to the CNS can cause neuronal injury by at least two processes, apoptosis and necrosis. Apoptosis is generally an active process in which the cells synthesize the proteins that result in their own demize (Villa et al. 1994). In contrast, necrosis does not appear to be dependent on protein synthesis and instead seems to be a passive process brought about by the inability to maintain ionic gradients and subsequent loss of homeostasis. Recent reports suggest that both apoptotic and non apoptotic modes of cell death can contribute to the damage that results from cerebral ischemia (Dizdaroglu 1992; Floyd and Carney 1992; Buettner 1993), trauma (Rink et al. 1995), and exposure to glutamate receptor agonists (Pollard et al. 1994; Gillardon et al. 1995) or beta-amyloid (Loo et al. 1993). Why insults lead to apoptotic death under some conditions and induce death through non apoptotic mechan- isms, such as necrosis, under other conditions has not been fully established, but the severity of damage seems to play a key role in determining the mode of cell death. For example, activation of the NMDA subtype of glutamate receptor is generally considered to cause necrosis (Fix et al. 1993; Ingvar et al. 1994). However, recent studies examining the response in greater detail suggest that low levels of NMDA activation and free radicals appear to induce apoptosis, whereas greater exposure to NMDA results in necrosis (Ankarcrona et al. 1995; Bonfoco et al. 1995). Furthermore, severe cerebral ischemia produces an acute necrotic response with less severe insults resulting in delayed apoptosis (Du et al. 1996). It is unclear whether stimuli that can cause both apoptosis and necrosis do so by damaging the same molecular target and how that damage might come about. A commonality to the aforementioned cerebral insults is that they involve oxidative stress, and free radicals contribute to the injury (Chan et al. 1987; Chan et al. 1990; Kumar et al. 1994; 520 q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 520±531 Received August 2, 2000; accepted September 7, 2000. Address correspondence and reprint requests to Glenn T. Gobbel, Department of Neurological Surgery, 200 Lothrop Street, Suite B400, PUH, University of Pittsburgh, Pittsburgh, PA 15238, USA. E-mail: gobbel@neuronet.pitt.edu Abbreviations used: DMSO, dimethyl sulfoxide; LDH, lactate dehydrogenase; MDA, malonaldehyde bis(dimethyl acetate); MEM, modi®ed Eagle's medium; PBS, phosphate-buffered saline.