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.
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.
Perinatal asphyxia induces neuronal cell death and brain injury, and is often associated with irreversible neurological deficits in children. There is an urgent need to elucidate the neuronal death ...mechanisms occurring after neonatal hypoxia-ischemia (HI). We here investigated the selective neuronal deletion of the Atg7 (autophagy related 7) gene on neuronal cell death and brain injury in a mouse model of severe neonatal hypoxia-ischemia. Neuronal deletion of Atg7 prevented HI-induced autophagy, resulted in 42% decrease of tissue loss compared to wild-type mice after the insult, and reduced cell death in multiple brain regions, including apoptosis, as shown by decreased caspase-dependent and -independent cell death. Moreover, we investigated the lentiform nucleus of human newborns who died after severe perinatal asphyxia and found increased neuronal autophagy after severe hypoxic-ischemic encephalopathy compared to control uninjured brains, as indicated by the numbers of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3)-, LAMP1 (lysosomal-associated membrane protein 1)-, and CTSD (cathepsin D)-positive cells. These findings reveal that selective neuronal deletion of Atg7 is strongly protective against neuronal death and overall brain injury occurring after HI and suggest that inhibition of HI-enhanced autophagy should be considered as a potential therapeutic target for the treatment of human newborns developing severe hypoxic-ischemic encephalopathy.
Macroautophagy (hereafter called autophagy) is an essential physiological process of degradation of organelles and long-lived proteins. The discovery of autosis, a Na
/K
-ATPase (ATP1)-dependent type ...of autophagic cell death with specific morphological and biochemical features, has strongly contributed to the acceptance of a pro-death role of autophagy. However, the occurrence and relevance of autosis in neurons has never been clearly investigated, whereas we previously provided evidence that autophagy mechanisms could be involved in neuronal death in different in vitro and in vivo rodent models of hypoxia-ischemia (HI) and that morphological features of autosis were observed in dying neurons following rat perinatal cerebral HI. In the present study, we demonstrated that neuronal autosis could occur in primary cortical neurons using two different stimulations enhancing autophagy flux and neuronal death: a neurotoxic concentration of Tat-BECN1 (an autophagy-inducing peptide) and a hypoxic/excitotoxic stimulus (mimicking neuronal death induced by cerebral HI). Both stimulations induce autophagic neuronal death (dependent on canonical autophagic genes and independent on apoptotic, necroptotic or ferroptotic pathways) with all morphological and biochemical (ATP1a-dependent) features of autosis. However, we demonstrated that autosis is not dependent on the ubiquitous subunit ATP1a1 in neurons, as in dividing cell types, but on the neuronal specific ATP1a3 subunit. We also provided evidence that, in different in vitro and in vivo models where autosis is induced, ATP1a3-BECN1 interaction is increased and prevented by cardiac glycosides treatment. Interestingly, an increase in ATP1a3-BECN1 interaction is also detected in dying neurons in the autoptic brains of human newborns with severe hypoxic-ischemic encephalopathy (HIE). Altogether, these results suggest that ATP1a3-BECN1-dependent autosis could play an important role in neuronal death in HI conditions, paving the way for the development of new neuroprotective strategies in hypoxic-ischemic conditions including in severe case of human HIE.
Lactate can be used by neurons as an energy substrate to support their activity. Evidence suggests that lactate also acts on a metabotropic receptor called HCAR1, first described in the adipose ...tissue. Whether HCAR1 also modulates neuronal circuits remains unclear. In this study, using qRT-PCR, we show that HCAR1 is present in the human brain of epileptic patients who underwent resective surgery. In brain slices from these patients, pharmacological HCAR1 activation using a non-metabolized agonist decreased the frequency of both spontaneous neuronal Ca2+ spiking and excitatory post-synaptic currents (sEPSCs). In mouse brains, we found HCAR1 expression in different regions using a fluorescent reporter mouse line and in situ hybridization. In the dentate gyrus, HCAR1 is mainly present in mossy cells, key players in the hippocampal excitatory circuitry and known to be involved in temporal lobe epilepsy. By using whole-cell patch clamp recordings in mouse and rat slices, we found that HCAR1 activation causes a decrease in excitability, sEPSCs, and miniature EPSCs frequency of granule cells, the main output of mossy cells. Overall, we propose that lactate can be considered a neuromodulator decreasing synaptic activity in human and rodent brains, which makes HCAR1 an attractive target for the treatment of epilepsy.
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.
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 β-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.
Despite tremendous advances in neonatal intensive care over the past 20 years, prematurity carries a high burden of neurological morbidity lasting lifelong. The term encephalopathy of prematurity ...(EoP) coined by Volpe in 2009 encompasses all aspects of the now known effects of prematurity on the immature brain, including altered and disturbed development as well as specific lesional hallmarks. Understanding the way cells are damaged is crucial to design brain protective strategies, and in this purpose, preclinical models largely contribute to improve the comprehension of the cell death mechanisms. While neuronal cell death has been deeply investigated and characterized in (hypoxic-ischemic) encephalopathy of the newborn at term, little is known about the types of cell death occurring in preterm brain injury. Three main different morphological cell death types are observed in the immature brain, specifically in models of hypoxic-ischemic encephalopathy, namely, necrotic, apoptotic, and autophagic cell death. Features of all three types may be present in the same dying neuron. In preterm brain injury, description of cell death types is sparse, and cell loss primarily concerns immature oligodendrocytes and, infrequently, neurons. In the present review, we first shortly discuss the different main severe preterm brain injury conditions that have been reported to involve cell death, including periventricular leucomalacia (PVL), diffuse white matter injury (dWMI), and intraventricular hemorrhages, as well as potentially harmful iatrogenic conditions linked to premature birth (anesthesia and caffeine therapy). Then, we present an overview of current evidence concerning cell death in both clinical human tissue data and preclinical models by focusing on studies investigating the presence of cell death allowing discriminating between the types of cell death involved. We conclude that, to improve brain protective strategies, not only apoptosis but also other cell death (such as regulated necrotic and autophagic) pathways now need to be investigated together in order to consider all cell death mechanisms involved in the pathogenesis of preterm brain damage.
Neuronal nitric oxide synthase (nNOS) and p38MAPK are strongly implicated in excitotoxicity, a mechanism common to many neurodegenerative conditions, but the intermediary mechanism is unclear. NOS1AP ...is encoded by a gene recently associated with sudden cardiac death, diabetes-associated complications, and schizophrenia (Arking et al., 2006; Becker et al., 2008; Brzustowicz, 2008; Lehtinen et al., 2008). Here we find it interacts with p38MAPK-activating kinase MKK3. Excitotoxic stimulus induces recruitment of NOS1AP to nNOS in rat cortical neuron culture. Excitotoxic activation of p38MAPK and subsequent neuronal death are reduced by competing with the nNOS:NOS1AP interaction and by knockdown with NOS1AP-targeting siRNAs. We designed a cell-permeable peptide that competes for the unique PDZ domain of nNOS that interacts with NOS1AP. This peptide inhibits NMDA-induced recruitment of NOS1AP to nNOS and in vivo in rat, doubles surviving tissue in a severe model of neonatal hypoxia-ischemia, a major cause of neonatal death and pediatric disability. The highly unusual sequence specificity of the nNOS:NOS1AP interaction and involvement in excitotoxic signaling may provide future opportunities for generation of neuroprotectants with high specificity.
Objective
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.
Methods
Neuronal autophagy and cell death were evaluated in the thalamus (frequently injured in severe HIE) of both human newborns who died after severe HIE (n = 5) and P7 hypoxic–ischemic rats (Rice–Vannuci model). Autophagic (LC3, p62), lysosomal (LAMP1, cathepsins), and cell death (TUNEL, caspase‐3) markers were studied by immunohistochemistry in human and rat brain sections, and by additional methods in rats (immunoblotting, histochemistry, and electron microscopy).
Results
Following severe perinatal asphyxia in both humans and rats, thalamic neurons displayed up to 10‐fold (p < 0.001) higher numbers of autophagosomes and lysosomes, implying an enhanced autophagic flux. The highly autophagic neurons presented strong features of apoptosis. These findings were confirmed and elucidated in more detail in rats.
Interpretation
These results show for the first time that autophagy is enhanced in severe HIE in dying thalamic neurons of human newborns, as in rats. Experimental neuroprotective strategies targeting autophagy could thus be a promising lead to follow for the development of future therapeutic approaches. Ann Neurol 2014;76:695–711
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