The evolutionarily conserved execution phase of apoptosis is defined by characteristic changes occurring during the final stages of death; specifically cell shrinkage, dynamic membrane blebbing, condensation of chromatin, and DNA fragmentation. Mechanisms underlying these hallmark features of apoptosis have previously been elusive, largely because the execution phase is a rapid event whose onset is asynchronous across a population of cells. In the present study, a model system is described for using the caspase inhibitor, z-VAD-FMK, to block apoptosis and generate a synchronous population of cells actively extruding and retracting membrane blebs. This model system allowed us to determine signaling mechanisms underlying this characteristic feature of apoptosis. A screen of kinase inhibitors performed on synchronized blebbing cells indicated that only myosin light chain kinase (MLCK) inhibitors decreased blebbing. Immunoprecipitation of myosin II demonstrated that myosin regulatory light chain (MLC) phosphorylation was increased in blebbing cells and that MLC phosphorylation was prevented by inhibitors of MLCK. MLC phosphorylation is also mediated by the small G protein, Rho. C3 transferase inhibited apoptotic membrane blebbing, supporting a role for a Rho family member in this process. Finally, blebbing was also inhibited by disruption of the actin cytoskeleton. Based on these results, a working model is proposed for how actin/myosin II interactions cause cell contraction and membrane blebbing. Our results provide the first evidence that MLC phosphorylation is critical for apoptotic membrane blebbing and also implicate Rho signaling in these active morphological changes. The model system described here should facilitate future studies of MLCK, Rho, and other signal transduction pathways activated during the execution phase of apoptosis.

Sublethal insults can induce tolerance to subsequent stressors in neurons. As cell death activators such as ROS generation and decreased ATP can initiate tolerance, we tested whether other cellular elements normally associated with neuronal injury could add to this process. In an in vivo model of ischemic tolerance, we were surprised to observe widespread caspase 3 cleavage, without cell death, in preconditioned tissue. To dissect the preconditioning pathways activating caspases, and the mechanisms by which these proteases are held in check, we developed an in vitro model of excitotoxic tolerance. In this model, antioxidants and caspase inhibitors blocked ischemia-induced protection against N-methyl-D-aspartate toxicity. Moreover, agents that blocked preconditioning also attenuated induction of HSP 70; transient overexpression of a constitutive form of this protein prevented HSP 70 upregulation and blocked tolerance. We outline a neuroprotective pathway where events normally associated with apoptotic cell death are critical for cell survival. P rior exposure to sublethal challenges can render neuronal tissue less vulnerable to severe insults (1). Preconditioning models share several key features, including limited window of efficacy, requirement for protein synthesis, involvement of ATP sensitive K ϩ (K ATP ) channels, and heat-shock protein (HSP) induction (2, 3). However, the underlying mechanisms mediating neuroprotection remain undefined. The up-regulation of prosurvival elements within preconditioned cells seems to depend upon activation of pathways typically associated with degeneration. For example, generation of reactive oxygen species (ROS) is critical for induction of tolerance in cardomyocytes (4, 5) and neurons (6-9). Metabolic dysfunction also contributes to preconditioning, as decline in ATP͞ADP ratios leads to mitochondrial K ATP channel opening (8) and ROS production (10). In fact, neuronal preconditioning is attenuated with K ATP antagonists (2), and K ATP activators are neuroprotective (11).Although ROS and energetic dysfunction contribute to preconditioning, little is known about how far these pathways progress before being halted, or the mechanism by which they are blocked. Here, we investigated the extent of activation of cell death pathways during ischemic preconditioning (IP) in vivo as well as the mechanism by which activation of these pathways results in tolerance in vitro. We propose a new model of IP in which neuroprotection depends upon activation of factors typically associated with neurodegeneration. Materials and MethodsFocal IP, Immunohistochemistry, and Immunoblotting. Transient MCAO was performed on spontaneously hypertensive rats (12). Immunohistochemistry for activated caspase 3 was performed as described (12). At various times after preconditioning, tissue was harvested, and proteins were run by SDS͞PAGE (13). For details, see Supporting Materials and Methods, which is published as supporting information on the PNAS web site, www.pnas.org.Tissue Culture and in Vitro Precondit...

Oxidant-induced neuronal apoptosis has been shown to involve potassium and zinc dysregulation, energetic dysfunction, activation of stress-related kinases, and caspase cleavage. The temporal ordering and interdependence of these events was investigated in primary neuronal cultures exposed to the sulfhydryl oxidizing agent 2,2Ј-dithiodipyridine (DTDP), a compound that induces the intracellular release of zinc. We previously observed that tetraethylammonium (TEA), high extracellular potassium, or cysteine protease inhibitors block apoptosis induced by DTDP. We now report that both p38 and extracellular signal-regulated kinase phosphorylation are evident in neuronal cultures within 2 hr of a brief exposure to 100 M DTDP. However, only p38 inhibition is capable of blocking oxidant-induced toxicity. Cyclohexamide or actinomycin D does not attenuate DTDPinduced cell death, suggesting that posttranslational modification of existing targets, rather than transcriptional activation, is responsible for the deleterious effects of p38. Indeed, an early robust increase in TEA-sensitive potassium channel currents induced by DTDP is attenuated by p38 inhibition but not by caspase inhibition. Moreover, we found that activation of p38 is required for caspase 3 and 9 cleavage, suggesting that potassium currents enhancement is required for caspase activation. Finally, we observed that DTDP toxicity could be blocked with niacinamide or benzamide, inhibitors of poly (ADP-ribose) synthetase. Based on these findings, we conclude that oxidation of sulfhydryl groups on intracellular targets results in intracellular zinc release, p38 phosphorylation, enhancement of potassium currents, caspase cleavage, energetic dysfunction, and translationally independent apoptotic cell death.

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