Vanessa Ginet

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.

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.

Enhancement of Autophagic Flux after Neonatal Cerebral Hypoxia-Ischemia and Its Region-Specific Relationship to Apoptotic Mechanisms

The multiplicity of cell death mechanisms induced by neonatal hypoxia-ischemia makes neuroprotective treatment against neonatal asphyxia more difficult to achieve. Whereas the roles of apoptosis and necrosis in such conditions have been studied intensively, the implication of autophagic cell death has only recently been considered. Here, we used the most clinically relevant rodent model of perinatal asphyxia to investigate the involvement of autophagy in hypoxic-ischemic brain injury. Seven-day-old rats underwent permanent ligation of the right common carotid artery, followed by 2 hours of hypoxia. This condition not only increased autophagosomal abundance (increase in microtubule-associated protein 1 light chain 3-11 level and punctuate labeling) but also lysosomal activities (cathepsin D, acid phosphatase, and beta-N-acetylhexosaminidase) in cortical and hippocampal CA3-damaged neurons at 6 and 24 hours, demonstrating an increase in the autophagic flux. In the cortex, this enhanced autophagy may be related to apoptosis since some neurons presenting a high level of autophagy also expressed apoptotic features, including cleaved caspase-3. On the other hand, enhanced autophagy in CA3 was associated with a more purely autophagic cell death phenotype. In striking contrast to CA3 neurons, those in CA1 presented only a minimal increase in autophagy but strong apoptotic characteristics. These results suggest a role of enhanced autophagy in delayed neuronal death after severe hypoxia-ischemia that is differentially linked to apoptosis according to the cerebral region.

Multiple interacting cell death mechanisms in the mediation of excitotoxicity and ischemic brain damage: a challenge for neuroprotection.

There is currently no approved neuroprotective pharmacotherapy for acute conditions such as stroke and cerebral asphyxia. One of the reasons for this may be the multiplicity of cell death mechanisms, because inhibition of a particular mechanism leaves the brain vulnerable to alternative ones. It is therefore essential to understand the different cell death mechanisms and their interactions. We here review the multiple signaling pathways underlying each of the three main morphological types of cell death--apoptosis, autophagic cell death and necrosis--emphasizing their importance in the neuronal death that occurs during cerebral ischemia and hypoxia-ischemia, and we analyze the interactions between the different mechanisms. Finally, we discuss the implications of the multiplicity of cell death mechanisms for the design of neuroprotective strategies.

Involvement of autophagy in hypoxic-excitotoxic neuronal death

Neuronal autophagy is increased in numerous excitotoxic conditions including neonatal cerebral hypoxia-ischemia (HI). However, the role of this HI-induced autophagy remains unclear. To clarify this role we established an in vitro model of excitotoxicity combining kainate treatment (Ka, 30 µM) with hypoxia (Hx, 6% oxygen) in primary neuron cultures. KaHx rapidly induced excitotoxic death that was completely prevented by MK801 or EGTA. KaHx also stimulated neuronal autophagic flux as shown by a rise in autophagosome number (increased levels of LC3-II and punctate LC3 labeling) accompanied by increases in lysosomal abundance and activity (increased SQSTM1/p62 degradation, and increased LC3-II levels in the presence of lysosomal inhibitors) and fusion (shown using an RFP-GFP-LC3 reporter). To determine the role of the enhanced autophagy we applied either pharmacological autophagy inhibitors (3-methyladenine or pepstatinA/E64) or lentiviral vectors delivering shRNAs targeting Becn1 or Atg7. Both strategies reduced KaHx-induced neuronal death. A prodeath role of autophagy was also confirmed by the enhanced toxicity of KaHx in cultures overexpressing BECN1 or ATG7. Finally, in vivo inhibition of autophagy by intrastriatal injection of a lentiviral vector expressing a Becn1-targeting shRNA increased the volume of intact striatum in a rat model of severe neonatal cerebral HI. These results clearly show a death-mediating role of autophagy in hypoxic-excitotoxic conditions and suggest that inhibition of autophagy should be considered as a neuroprotective strategy in HI brain injuries.

Mfn2 downregulation in excitotoxicity causes mitochondrial dysfunction and delayed neuronal death.

Mitochondrial fusion and fission is a dynamic process critical for the maintenance of mitochondrial function and cell viability. During excitotoxicity neuronal mitochondria are fragmented, but the mechanism underlying this process is poorly understood. Here, we show that Mfn2 is the only member of the mitochondrial fusion/fission machinery whose expression is reduced in in vitro and in vivo models of excitotoxicity. Whereas in cortical primary cultures, Drp1 recruitment to mitochondria plays a primordial role in mitochondrial fragmentation in an early phase that can be reversed once the insult has ceased, Mfn2 downregulation intervenes in a delayed mitochondrial fragmentation phase that progresses even when the insult has ceased. Downregulation of Mfn2 causes mitochondrial dysfunction, altered calcium homeostasis, and enhanced Bax translocation to mitochondria, resulting in delayed neuronal death. We found that transcription factor MEF2 regulates basal Mfn2 expression in neurons and that excitotoxicity‐dependent degradation of MEF2 causes Mfn2 downregulation. Thus, Mfn2 reduction is a late event in excitotoxicity and its targeting may help to reduce excitotoxic damage and increase the currently short therapeutic window in stroke.

Limited role of the c-Jun N-terminal kinase pathway in a neonatal rat model of cerebral hypoxia-ischemia.

D‐JNKI1, a cell‐permeable peptide inhibitor of the c‐Jun N‐terminal kinase (JNK) pathway, has been shown to be a powerful neuroprotective agent after focal cerebral ischemia in adult mice and young rats. We have investigated the potential neuroprotective effect of D‐JNKI1 and the involvement of the JNK pathway in a neonatal rat model of cerebral hypoxia–ischemia (HI). Seven‐day‐old rats underwent a permanent ligation of the right common carotid artery followed by 2 h of hypoxia (8% oxygen). Treatment with D‐JNKI1 (0.3 mg/kg intraperitoneally) significantly reduced early calpain activation, late caspase 3 activation and, in the thalamus, autophagosome formation, indicating an involvement of JNK in different types of cell death: necrotic, apoptotic, and autophagic. However, the size of the lesion was unchanged. Further analysis showed that neonatal HI induced an immediate decrease in JNK phosphorylation (reflecting mainly JNK1 phosphorylation) followed by a slow progressive increase (including JNK3 phosphorylation 54 kDa), whereas c‐jun and c‐fos expression were both strongly activated immediately after HI. In conclusion, unlike in adult ischemic models, JNK is only moderately activated after severe cerebral HI in neonatal rats and the observed positive effects of D‐JNKI1 are insufficient to give neuroprotection. Thus, for perinatal asphyxia, D‐JNKI1 can only be considered in association with other therapies.

Neuronal death after perinatal cerebral hypoxia-ischemia: Focus on autophagy-mediated cell death.

Neonatal hypoxic‐ischemic encephalopathy is a critical cerebral event occurring around birth with high mortality and neurological morbidity associated with long‐term invalidating sequelae. In view of the great clinical importance of this condition and the lack of very efficacious neuroprotective strategies, it is urgent to better understand the different cell death mechanisms involved with the ultimate aim of developing new therapeutic approaches. The morphological features of three different cell death types can be observed in models of perinatal cerebral hypoxia‐ischemia: necrotic, apoptotic and autophagic cell death. They may be combined in the same dying neuron. In the present review, we discuss the different cell death mechanisms involved in neonatal cerebral hypoxia‐ischemia with a special focus on how autophagy may be involved in neuronal death, based: (1) on experimental models of perinatal hypoxia‐ischemia and stroke, and (2) on the brains of human neonates who suffered from neonatal hypoxia‐ischemia.

Dying neurons in thalamus of asphyxiated term newborns and rats are autophagic

Neonatal hypoxic–ischemic encephalopathy (HIE) still carries a high burden by its mortality and long‐term neurological morbidity in survivors. Apart from hypothermia, there is no acknowledged therapy for HIE, reflecting the lack of mechanistic understanding of its pathophysiology. (Macro)autophagy, a physiological intracellular process of lysosomal degradation, has been proposed to be excessively activated in excitotoxic conditions such as HIE. The present study examines whether neuronal autophagy in the thalamus of asphyxiated human newborns or P7 rats is enhanced and related to neuronal death processes.

Autophagy Defect Is Associated with Low Glucose-Induced Apoptosis in 661W Photoreceptor Cells

Glucose is an important metabolic substrate of the retina and diabetic patients have to maintain a strict normoglycemia to avoid diabetes secondary effects, including cardiovascular disease, nephropathy, neuropathy and retinopathy. Others and we recently demonstrated the potential role of hypoglycemia in diabetic retinopathy. We showed acute hypoglycemia to induce retinal cell death both in vivo during an hyperinsulinemic/hypoglycemic clamp and in vitro in 661W photoreceptor cells cultured at low glucose concentration. In the present study, we showed low glucose to induce a decrease of BCL2 and BCL-XL anti-apoptotic proteins expression, leading to an increase of free pro-apoptotic BAX. In parallel, we showed that, in retinal cells, low glucose-induced apoptosis is involved in the process of autophagosomes formation through the AMPK/RAPTOR/mTOR pathway. Moreover, the decrease of LAMP2a expression led to a defect in the autophagosome/lysosome fusion process. Specific inhibition of autophagy, either by 3-methyladenine or by down-regulation of ATG5 or ATG7 proteins expression, increased caspase 3 activation and 661W cell death. We show that low glucose modifies the delicate equilibrium between apoptosis and autophagy. Cells struggled against low nutrient condition-induced apoptosis by starting an autophagic process, which led to cell death when inhibited. We conclude that autophagy defect is associated with low glucose-induced 661W cells death that could play a role in diabetic retinopathy. These results could modify the way of addressing negative effects of hypoglycemia. Short-term modulation of autophagy could be envisioned to treat diabetic patients in order to avoid secondary complications of the disease.