The term "programmed cell death," and its synonym, "apoptosis," both invoke a vision of an orderly dismantling and disposal of the machinery of a living cell. Regulated by a cascade of caspase proteases, apoptosis is the dignified way for cells to die. In contrast, hear the word "necrosis" and the image that comes to mind is more like that of a train wreck. Necrosis is a far messier and unregulated affair where cells precipitously fall apart in the face of overwhelming insult. While necrosis and apoptosis both play important roles in the death of neurons—in stroke and neurodegenerative disease, for example—apoptosis has received the lion’s share of attention because it is considered a therapeutically tractable pathway (see Alzforum live discussion and Yuan et al., 2004). With the report of a new regulated cell death program, that may be about to change.

Junying Yuan and collaborators at Harvard Medical School have dubbed the caspase-independent, non-apoptotic, alternative death program “necroptosis,” because it shares many features with necrosis—including cell morphology changes, mitochondrial dysfunction, loss of plasma membrane integrity, and autophagic clearance of cell debris—and because it is triggered through the same family of death receptors that control apoptosis. A demonstration that a small molecule inhibitor of necroptosis can block neuron death in a mouse model of ischemia at once proves the physiological relevance of the alterative pathway and offers a new prospect for treating stroke. The availability of the inhibitor will also speed the task of determining the potential role of necroptosis in other pathologies, including Alzheimer disease, where death receptors have been implicated in amyloid-β toxicity (see ARF related news story and Li et al., 2004).

The work, reported in the May 29 online edition of Nature Chemical Biology, was stimulated by the observation that caspase inhibitors, despite their ability to potently and specifically block apoptosis, cannot always save cells from dying. Reports that activation of the Fas/tumor necrosis factor receptor (TNFR) family of death receptors caused necrosis even in the presence of caspase inhibitors led Yuan and coworkers to ask if there was another death program activated by the receptors. To investigate this, first author Alexei Degterev and his colleagues screened a chemical library of 15,000 compounds for inhibitors that could prevent the death of human U937 cells treated with TNF and a caspase inhibitor. From the screen, the authors isolated a compound they called necrostatin-1 (Nec-1), which inhibited necroptosis—cell death induced by death receptors but independent of caspase activation—in several cell types. The authors found that necrostatin could also prevent the death of cells lacking the signaling molecule FADD, which is essential for caspase activation. Nec-1 specifically prevented the mitochondrial dysfunction, loss of cellular ATP content and plasma membrane integrity, and late-stage autophagy response, which are all absent in apoptosis, but it had no effect on healthy cells or those undergoing caspase-dependent apoptosis.

To look for a physiologic role of necroptosis, the researchers turned to stroke, an injury where non-apoptotic cell death is known to be important. When Degterev and colleagues administered Nec-1 to mice, it significantly, and in a dose-dependent manner, reduced the infarct volume caused by subsequent blockage of the cerebral artery. Following the trauma, Nec-1-treated mice also had much more improvement in neurologic function compared to controls. Nec-1 did not block caspase activation, and its protective effect was additive with caspase inhibitors. However, unlike the latter, which must be given within a few hours of the trauma to have an effect, Nec-1 still worked when administration was delayed up to 6 hours, consistent with the observation of a delayed activation of necroptosis. For stroke, the extended time window for therapy would be a valuable asset.

The actions of Nec-1 in vitro and in vivo raise some immediate questions. First, what is the target of Nec-1? Structure-activity studies suggested that Nec-1 binds to a specific target, but the protein or pathway is so far unknown. Second, what are the triggers for necroptosis and what is its physiological role in relation to apoptosis? In non-neuronal cells, necroptosis may serve as a back-up mechanism to eliminate cells when apoptosis fails, speculates Yuan. But during brain ischemia in vivo, necroptosis appeared to play a primary role in neuronal death. “We hypothesize that intrinsic heterogeneity of neuronal populations and, potentially, conditions of stress might render some neuronal cells too ‘damaged’ to undergo ordered apoptosis, making them undergo necroptotic death instead,” Yuan explained. “We are currently in the process of characterizing induction of necroptosis in response to various neurodegenerative stimuli in vitro; however, already available in vivo data for the necrostatin-mediated neuroprotection following brain ischemia suggest that the necroptosis pathway is expressed in neurons and might play an important role in neuronal pathologic death, making necrostatins an interesting new class of neuroprotective agents.”—Pat McCaffrey.

Reference:
Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, Cuny GD, Mitchison TJ, Moskowitz MA, Yuan J. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nature Chemical Biology. 29 May 2005. Advanced online publication. Abstract

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  1. The paper by Degterev et al. is a tour de force characterization of necroptosis, a non-apoptotic form of programmed cell death that involves necrosis and autophagy. Existence of this caspase-independent pathway was hypothesized from observations that several different cultured cell types undergo a common necrotic death upon stimulation of death domain receptor proteins in the presence of caspase inhibitors. To investigate a necroptosis pathway, the authors performed a chemical screen of 15,000 small molecules for necroptosis inhibition. Of these, a heterocyclic compound, Necrostatin-1 (Nec-1), was shown to be a very potent and specific inhibitor of necroptosis.

    Application of Nec-1 did not block apoptosis, autophagy, or oxidative stress-induced necrosis, and also did not disrupt normal cellular physiology. Significantly, necroptosis was shown to be a delayed component of ischemia-associated neuronal cell death induced by cerebral artery occlusion in mice. Administration of Nec-1 attenuated the extent of ischemia-induced neuronal death and did not disrupt general brain physiology. Furthermore, Nec-1 exhibited an extended time window of protection and was able to exert its effects 6 hours after the onset of injury. The simultaneous addition of Nec-1 and the zVAD.fmk caspase inhibitor yielded an additive protective effect, suggesting a potentially effective therapeutic combination.

    Overall, necroptosis has a delayed latency compared to apoptosis, and the authors hypothesize that it may act as a redundant mechanism to provide cells with an ability to die when they find themselves in an environment non-permissive to apoptosis. Future studies to determine the site of Nec-1 action and characterize the components of necroptosis pathway promise to provide important insight not only into a conserved and important mechanism for cell death, but also to develop effective treatments for a variety of human pathologies.

  2. Two general mechanisms of cell death have been described: programmed cell death and necrosis. Programmed cell death, or apoptosis, is a directed program that proceeds through specific signal transduction pathways common to different cell types. In particular, apoptosis initiates a sequential activation of multiple caspases. In contrast, the alternative to programmed cell death, necrosis, is thought to be a nondirected cellular response to overwhelming stress. Therapeutic strategies to prevent cell death in pathological conditions have targeted apoptosis rather than necrosis, because of the perception that necrosis is unregulated and relatively nonspecific. However, recent reports have implicated specific signal transduction pathways, such as stimulation of death domain receptors (DRs) by their ligands, in necrotic cell death. In a paper that is stunning in its elegance and simplicity, Degterev et al. build on these observations by identifying a new type of programmed cell death that resembles necrosis but is distinct from both apoptosis and necrosis. They call it necroptosis.

    The authors had followed the growing number of studies suggesting that under certain situations, DR-induced cell death, which normally proceeds via an apoptotic pathway, is not prevented by caspase inhibitors and resembles necrosis. Because this caspase-independent DR-induced cell death led to similar necrotic morphological features in a wide variety of cell types, Degterev and collaborators suspected the involvement of a non-apoptotic programmed signal transduction pathway shared by multiple cell types. They chose an ingenious way to find out whether such a pathway actually exists. Cells treated simultaneously with the DR agonist TNFα and a pan-caspase inhibitor, a combination the authors used to devise an operational definition of necroptosis, were used to screen a library of chemical compounds for inhibitors of the death of these cells. The screen resulted in the selection of a molecule dubbed necrostatin-1 (Nec-1).

    The authors then used Nec-1 to answer a number of questions about this new pathway that they called necroptosis. First, they asked whether this pathway was indeed distinct from apoptosis. When cells are exposed to FasL (Fas ligand), they exhibit classic symptoms of apoptosis. Stimulation of cells with FasL in the presence of a pan-caspase inhibitor, in contrast, leads to morphological symptoms of necrosis. The authors showed that Nec-1 did not inhibit apoptotic morphology (cytoplasm condensation, chromatin marginalization, nuclear fragmentation, and plasma membrane blebbing) displayed by FasL-treated cells. However, Nec-1 did inhibit the appearance of necrotic morphology (nuclear condensation, organelle swelling, and early loss of plasma membrane integrity) displayed by cells exposed to FasL in the presence of the caspase inhibitor zVAD.fmk. Of special interest was the fact that the onset of apoptosis in response to FasL was faster than the onset of necroptosis in response to FasL in conjunction with zVAD.fmk. The authors suggest that apoptosis usually conceals or forestalls necroptosis because of its faster kinetics.

    The authors then asked whether necroptotic cell death utilized factors involved in known cell death signaling pathways. They compared the activity of Nec-1 with that of small-molecule inhibitors of such factors as calpains, calcium homeostasis perturbation, PARP, and nitric oxide synthase. None of the tested compounds inhibited necroptosis in all cell types, as Nec-1 does, establishing the uniqueness of the necroptotic pathway. Furthermore, necroptosis was not inhibited by antioxidants, nor did Nec-1 block the classic necrosis caused by the cell stressor menadione, showing the independence of necroptosis from oxidative stress.

    Neuronal cell death caused by ischemic brain injury is known to display some non-apoptotic features, and the participation of DRs in ischemic cell death has been postulated. The authors thought that perhaps ischemia produces conditions that are more conducive to necroptosis than to apoptosis. So they administered Nec-1 intracerebroventricularly to mice that had undergone middle cerebral artery occlusion (MCAO), a model for inducing ischemic damage in mice. Strikingly, Nec-1 reduced the infarct volume without blocking caspase 3 activation, showing that at least a portion of the cell death resulting from MCAO is necroptotic.

    A growing body of evidence supports the idea that apoptosis is at least one means by which neurons die in Alzheimer disease (AD). However, a number of studies have described non-apoptotic features of AD neurodegeneration. Furthermore, DRs have been implicated both in neuritic degeneration in AD brain and in neuronal death induced by β-amyloid (e.g., Morishima et al., 2001). It is possible that necroptosis also plays a role in AD neurodegeneration. It will be interesting to assess the effect of Nec-1 on neuropathology in mouse transgenic models of AD, or on neurodegeneration in in vitro models of AD neurodegeneration. If necroptosis is shown to be a component of AD pathology, a new world of therapeutic strategies, aimed at necroptotic pathways, would be opened up for this devastating disease.

References

News Citations

  1. Dying to Bind: DENN/MADD Promotes Neuron Death in AD Brain

Paper Citations

  1. . Diversity in the mechanisms of neuronal cell death. Neuron. 2003 Oct 9;40(2):401-13. PubMed.
  2. . Tumor necrosis factor death receptor signaling cascade is required for amyloid-beta protein-induced neuron death. J Neurosci. 2004 Feb 18;24(7):1760-71. PubMed.
  3. . Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol. 2005 Jul;1(2):112-9. PubMed.

Other Citations

  1. Alzforum live discussion

Further Reading

Papers

  1. . Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol. 2005 Jul;1(2):112-9. PubMed.

Primary Papers

  1. . Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol. 2005 Jul;1(2):112-9. PubMed.