Peter G.H. Clarke

Developmental cell death : morphological diversity and multiple mechanisms

SummaryPhysiological cell death is a widespread phenomenon in the development of both vertebrates and invertebrates. This review concentrates on an aspect of developmental cell death that has tended to be neglected, the manner in which the cells are dismantled. It is emphasized that the dying cells may adopt one of at least three different morphological types: “apoptotic”, “autophagic”, and “non-lysosomal vesiculate”. These probably reflect a corresponding multiplicity of intracellular events. In particular, the destruction of the cytoplasm in these three types appears to be achieved primarily by heterophagy, by autophagy and by non-lysosomal degradation, respectively. The various mechanisms underlying both nuclear and cytoplasmic destruction are reviewed in detail. The multiplicity of destructive mechanisms needs to be born in mind in studies of other aspects of cell death such as the signals which trigger it, since different signals probably trigger different types of cell death.

A peptide inhibitor of c-Jun N-terminal kinase protects against excitotoxicity and cerebral ischemia

Neuronal death in cerebral ischemia is largely due to excitotoxic mechanisms, which are known to activate the c-Jun N-terminal kinase (JNK) pathway. We have evaluated the neuroprotective power of a cell-penetrating, protease-resistant peptide that blocks the access of JNK to many of its targets. We obtained strong protection in two models of middle cerebral artery occlusion (MCAO): transient occlusion in adult mice and permanent occlusion in 14-d-old rat pups. In the first model, intraventricular administration as late as 6 h after occlusion reduced the lesion volume by more than 90% for at least 14 d and prevented behavioral consequences. In the second model, systemic delivery reduced the lesion by 78% and 49% at 6 and 12 h after ischemia, respectively. Protection correlated with prevention of an increase in c-Jun activation and c-Fos transcription. In view of its potency and long therapeutic window, this protease-resistant peptide is a promising neuroprotective agent for stroke.

Autosis is a Na+,K+-ATPase–regulated form of cell death triggered by autophagy-inducing peptides, starvation, and hypoxia–ischemia

Significance We show that the selective overactivation of autophagy can cause cell death with unique morphological features distinct from apoptosis or necrosis. This unique type of autophagic cell death, termed “autosis,” occurs not only in vitro but also in vivo in cerebral hypoxia–ischemia. Moreover, autosis is inhibited both in vitro and in vivo by cardiac glycosides, which are Na+,K+-ATPase antagonists used in clinical medicine. Our findings contribute to the basic understanding of cell-death mechanisms and suggest strategies for protecting cells against stresses such as hypoxia–ischemia. A long-standing controversy is whether autophagy is a bona fide cause of mammalian cell death. We used a cell-penetrating autophagy-inducing peptide, Tat-Beclin 1, derived from the autophagy protein Beclin 1, to investigate whether high levels of autophagy result in cell death by autophagy. Here we show that Tat-Beclin 1 induces dose-dependent death that is blocked by pharmacological or genetic inhibition of autophagy, but not of apoptosis or necroptosis. This death, termed “autosis,” has unique morphological features, including increased autophagosomes/autolysosomes and nuclear convolution at early stages, and focal swelling of the perinuclear space at late stages. We also observed autotic death in cells during stress conditions, including in a subpopulation of nutrient-starved cells in vitro and in hippocampal neurons of neonatal rats subjected to cerebral hypoxia–ischemia in vivo. A chemical screen of ∼5,000 known bioactive compounds revealed that cardiac glycosides, antagonists of Na+,K+-ATPase, inhibit autotic cell death in vitro and in vivo. Furthermore, genetic knockdown of the Na+,K+-ATPase α1 subunit blocks peptide and starvation-induced autosis in vitro. Thus, we have identified a unique form of autophagy-dependent cell death, a Food and Drug Administration-approved class of compounds that inhibit such death, and a crucial role for Na+,K+-ATPase in its regulation. These findings have implications for understanding how cells die during certain stress conditions and how such cell death might be prevented.

Postischemic treatment of neonatal cerebral ischemia should target autophagy.

To evaluate the contributions of autophagic, necrotic, and apoptotic cell death mechanisms after neonatal cerebral ischemia and hence define the most appropriate neuroprotective approach for postischemic therapy.

Chapter 13 Neuron Death in Vertebrate Development: In Vivo Methods

Publisher Summary The study of neuronal death involves demonstrating that it occurs in a given situation, estimating the magnitude and timing of the loss, evaluating which particular neurons die, analyzing why and how they die, and understanding the role or purpose of the loss. A wide range of methods is available for achieving these ends; most involve the use of histological sections, although biochemical analysis of homogenized tissue can also provide useful information. Counting healthy neurons in histological sections is the most direct and widely used method for estimating the number of neurons that die, and the timing of their loss. Because most cases of neuronal death occur in postmitotic populations, there is rarely any need to consider complex tissue kinetics; the number of neurons lost is simply the initial number minus the final number in a defined population. However, the fact that subtraction is used makes the final estimation of neuronal death highly sensitive to errors in the estimations of total neuronal number. To meet the high standards of accuracy that are required in counting healthy neurons, it is essential to avoid two main sources of error: those because of inadequate definition of the population to be counted and those in counting.

NINETEENTH CENTURY RESEARCH ON NATURALLY OCCURRING CELL DEATH AND RELATED PHENOMENA

Research on naturally occurring cell death is older than current opinion gives credit. More than 100 nineteenth century publications deal with it, and we review most of these. Soon after the establishment of the cell theory by Schleiden and Schwann, Carl Vogt (1842) reported cell death in the notochord and adjacent cartilage of metamorphic toads. Subsequent landmark discoveries included the massive cell death that occurs in pupating diptera (Weismann 1864), chondrocyte death during endochondral ossification (Stieda 1872), phagocytosis associated with cell death in the muscles of metamorphic toads (Metschnikoff 1883), chromatolytic (apoptotic) cell death in ovarian follicles (Flemming 1885), the reinterpretation of “Sarkoplasten” as “Sarkolyten” in metamorphic amphibia (Mayer 1886), the programmed loss of an entire population of neurons in fish embryos (Beard 1889), the death of scattered myocytes and myofibres in mammalian muscle (Felix 1889), and the death of many motor and sensory neurons in chick embryos (Collin 1906). Other lines of nineteenth century research established concepts important for understanding cell death, notably trophic interactions between neurons and their targets, and intercellular competition.

N‐methyl‐d‐aspartate‐triggered neuronal death in organotypic hippocampal cultures is endocytic, autophagic and mediated by the c‐Jun N‐terminal kinase pathway

Acute excitotoxic neuronal death was studied in rat organotypic hippocampal slices exposed to 100 µmN‐methyl‐d‐aspartate. Fulgurant death of pyramidal neurons occurred in the CA1 and CA3 regions and was already detectable within 2 h of the N‐methyl‐d‐aspartate administration. Morphologically, the neuronal death was neither apoptotic nor necrotic but had the hallmarks of autophagic neuronal death, as shown by acid phosphatase histochemistry in both CA1 and CA3 and by electron microscopy in CA1. The dying neurons also manifested strong endocytosis of horseradish peroxidase or microperoxidase, occurring probably by a fluid phase mechanism, and followed, surprisingly, by nuclear entry. In addition to these autophagic and endocytic characteristics, there were indications that the c‐Jun N‐terminal kinase pathway was activated. Its target c‐Jun was selectively phosphorylated in CA1, CA3 and the dentate gyrus and c‐Fos, the transcription of which is under the positive control of c‐Jun N‐terminal kinase target Elk1, was selectively up‐regulated in CA1 and CA3. All these effects, the neuronal death itself and the associated autophagy and endocytosis, were totally prevented by a cell‐permeable inhibitor of the interaction between c‐Jun N‐terminal kinase and certain of its targets. These results show that pyramidal neurons undergoing excitotoxic death in this situation are autophagic and endocytic and that both the cell death and the associated autophagy and endocytosis are under the control of the c‐Jun N‐terminal kinase pathway.

Specific Targeting of Pro-Death NMDA Receptor Signals with Differing Reliance on the NR2B PDZ Ligand

NMDA receptors (NMDARs) mediate ischemic brain damage, for which interactions between the C termini of NR2 subunits and PDZ domain proteins within the NMDAR signaling complex (NSC) are emerging therapeutic targets. However, expression of NMDARs in a non-neuronal context, lacking many NSC components, can still induce cell death. Moreover, it is unclear whether targeting the NSC will impair NMDAR-dependent prosurvival and plasticity signaling. We show that the NMDAR can promote death signaling independently of the NR2 PDZ ligand, when expressed in non-neuronal cells lacking PSD-95 and neuronal nitric oxide synthase (nNOS), key PDZ proteins that mediate neuronal NMDAR excitotoxicity. However, in a non-neuronal context, the NMDAR promotes cell death solely via c-Jun N-terminal protein kinase (JNK), whereas NMDAR-dependent cortical neuronal death is promoted by both JNK and p38. NMDAR-dependent pro-death signaling via p38 relies on neuronal context, although death signaling by JNK, triggered by mitochondrial reactive oxygen species production, does not. NMDAR-dependent p38 activation in neurons is triggered by submembranous Ca2+, and is disrupted by NOS inhibitors and also a peptide mimicking the NR2B PDZ ligand (TAT-NR2B9c). TAT-NR2B9c reduced excitotoxic neuronal death and p38-mediated ischemic damage, without impairing an NMDAR-dependent plasticity model or prosurvival signaling to CREB or Akt. TAT-NR2B9c did not inhibit JNK activation, and synergized with JNK inhibitors to ameliorate severe excitotoxic neuronal loss in vitro and ischemic cortical damage in vivo. Thus, NMDAR-activated signals comprise pro-death pathways with differing requirements for PDZ protein interactions. These signals are amenable to selective inhibition, while sparing synaptic plasticity and prosurvival signaling.

Relationships between neuronal death and the cellular redox status. Focus on the developing nervous system.

During the development of the nervous system, a large number of neurons are eliminated through naturally occurring neuronal death. Many morphological and biochemical properties of such dying neurons are reminiscent of apoptosis, a type of death involving the action of genetically-programmed events but also epigenetic phenomena including oxidative stress. The following review contains three parts focusing respectively on basic knowledge of neuronal death and redox regulation, the mechanisms involved in neuronal death which are ordered in three sequential phases, and on the complex relations between neuronal fate and the redox status. Finally, we point out that oxidants are not always detrimental for neuronal survival. On the one hand, dying neurons often display signs of oxidative stress, including an elevation of their intracellular concentration of free radicals. Antioxidants may reduce the extent of neuronal death, suggesting a causal implication of free radicals in the death-process. On the other hand, at high concentrations antioxidants may lose their protective effects on developing neurons, and a non-lethal oxidative stress may potentiate the protective effects of other agents. These data suggest that free radicals, perhaps through their effects on cellular signalling pathways, may have positive effects on neuronal survival, provided that their intraneuronal concentrations are maintained at low levels. Much evidence suggests that the neuronal redox status must be maintained within a narrow range of values compatible with survival. Antioxidants may protect neurons subjected to an oxidative stress following axotomy or trophic factor-deprivation; but excessive reduction may become equally detrimental for neurons.

Beclin 1-independent autophagy contributes to apoptosis in cortical neurons

Neuronal autophagy is enhanced in many neurological conditions, such as cerebral ischemia and traumatic brain injury, but its role in associated neuronal death is controversial, especially under conditions of apoptosis. We therefore investigated the role of autophagy in the apoptosis of primary cortical neurons treated with the widely used and potent pro-apoptotic agent, staurosporine (STS). Even before apoptosis, STS enhanced autophagic flux, as shown by increases in autophagosomal (LC3-II level, LC3 punctate labeling) and lysosomal (cathepsin D, LAMP1, acid phosphatase, β-hexasominidase) markers. Inhibition of autophagy by 3-methyladenine, or by lentivirally-delivered shRNAs against Atg5 and Atg7, strongly reduced the STS-induced activation of caspase-3 and nuclear translocation of AIF, and gave partial protection against neuronal death. Pan-caspase inhibition with Q-VD-OPH likewise protected partially against neuronal death, but failed to affect autophagy. Combined inhibition of both autophagy and caspases gave strong synergistic neuroprotection. The autophagy contributing to apoptosis was Beclin 1-independent, as shown by the fact that Beclin 1 knockdown failed to reduce it but efficiently reduced rapamycin-induced autophagy. Moreover the Beclin 1 knockdown sensitized neurons to STS-induced apoptosis, indicating a cytoprotective role of Beclin 1 in cortical neurons. Caspase-3 activation and pyknosis induced by two other pro-apoptotic stimuli, MK801 and etoposide, were likewise found to be associated with Beclin 1-independent autophagy and reduced by the knockdown of Atg7 but not Beclin 1. In conclusion, Beclin 1-independent autophagy is an important contributor to both the caspase-dependent and -independent components of neuronal apoptosis and may be considered as an important therapeutic target in neural conditions involving apoptosis.