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.
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.
Cytomegalovirus is responsible for the most common congenital infection, affecting 0.5% to 1.0% of live births in Europe. Congenital cytomegalovirus infection can be diagnosed during pregnancy by ...viral DNA amplification in the amniotic fluid, but the prognosis of fetuses without severe brain abnormalities remains difficult to establish on the basis of prenatal imaging alone.
To identify predictors of moderate to severe symptomatic cytomegalovirus infection among fetal blood parameters and to propose an algorithm on the basis of these parameters and on prenatal imaging that would provide the best positive and negative predictive values.
Fetal blood sampling at 21–28 weeks gestation was performed in fetuses with congenital cytomegalovirus infection confirmed by amniocentesis after maternal infection in the first-trimester or periconceptional period. We compared the levels of hemoglobin, thrombocytes, γ-glutamyl transpeptidase, aspartate aminotransferase, alanine aminotransferase, β2-microglobulin, immunoglobulins G and M, and cytomegalovirus DNA viral loads in amniotic fluid and fetal blood between those with moderate to severe symptomatic infection and those with asymptomatic to mild infection (median follow-up of 36 months for live births).
Among 58 fetuses included, 25 (43%) had a moderate to severe symptomatic infection: 16 with severe cerebral abnormalities, 5 with multiple signs or symptoms at birth, 2 with bilateral sensorineural hearing loss, and 2 with neurodevelopmental delay. The values of thrombocytes, aspartate aminotransferase, β2 microglobulin, Immunoglobulin M, and cytomegalovirus viral loads differed significantly between fetuses with moderate to severe symptomatic infection and those with asymptomatic to mild infection. The optimal strategy to predict moderate to severe symptomatic infection was to first perform fetal brain imaging, followed by fetal blood sampling with the following cutoffs: thrombocytes <120,000/mL, viremia ≥5 log10/mL, and β2 microglobulin ≥12 mg/L). This recursive algorithm had a negative predictive value of 100% for moderately to severely symptomatic infection.
The combination of thrombocytes, β2-microglobulin, and cytomegalovirus viral load in fetal blood can be used for prognosis determination, particularly in cytomegalovirus-infected fetuses without severe brain abnormalities at the time of prenatal diagnosis. Future studies should evaluate whether these parameters remain useful in infected fetuses who have been treated with valacyclovir before fetal blood sampling.
Display omitted
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.
Objectives To investigate the association between early nutritional intake and brain development assessed by magnetic resonance imaging (MRI). Study design A cohort of neonates born at ≤30 weeks ...gestational age underwent MRI at term equivalent age. Brain maturation and injury were assessed using the Kidokoro score. Two groups were defined by severity of the scores. The associations between macronutrients intake during the first 2 weeks of life, clinical factors, and imaging scores were analyzed using logistic regression. Results MRI scores from group 1 patients (n = 27) were normal to mildly abnormal (0-5). Group 2 (n = 15) had more abnormal scores (6-12). The median gestational ages (IQR) were 27.4 (1.9) weeks in group 1 and 27.0 (2.9) weeks in group 2, with birth weights of 900 (318) g (group 1) and 844 (293) g (group 2). In group 2, energy, lipid, and carbohydrate intake were significantly lower than in group 1. Group 2 also showed higher rates of sepsis and clinical risk scores than group 1. After adjustments in bivariate models, higher energy and lipid intake remained significantly associated with improved scores on MRI. This association was stronger for the gray matter component of the score. Conclusions Higher energy and lipid intake during the first 2 weeks after birth was associated with a lower incidence of brain lesions and dysmaturation at term equivalent age in preterm neonates.
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.
Cystic periventricular leukomalacia is commonly diagnosed in premature infants, resulting from severe hypoxic-ischemic white matter injury, and also involving some grey matter damage. Very few is ...known concerning the cell death pathways involved in these types of premature cerebral lesions. Excitotoxicity is a predominant mechanism of hypoxic-ischemic injury in the developing brain. Concomitantly, it has been recently shown that autophagy could be enhanced in excitotoxic conditions switching this physiological intracellular degradation system to a deleterious process. We here investigated the role of autophagy in a validated rodent model of preterm excitotoxic brain damage mimicking in some aspects cystic periventricular leukomalacia. An excitotoxic lesion affecting periventricular white and grey matter was induced by injecting ibotenate, a glutamate analogue, in the subcortical white matter (subcingulum area) of five-day old rat pups. Ibotenate enhanced autophagy in rat brain dying neurons at 24 h as shown by increased presence of autophagosomes (increased LC3-II and LC3-positive dots) and enhanced autophagic degradation (SQSTM1 reduction and increased number and size of lysosomes (LAMP1- and CATHEPSIN B-positive vesicles)). Co-injection of the pharmacological autophagy inhibitor 3-methyladenine prevented not only autophagy induction but also CASPASE-3 activation and calpain-dependent cleavage of SPECTRIN 24 h after the insult, thus providing a strong reduction of the long term brain injury (16 days after ibotenate injection) including lateral ventricle dilatation, decreases in cerebral tissue volume and in subcortical white matter thickness. The autophagy-dependent neuroprotective effect of 3-methyladenine was confirmed in primary cortical neuronal cultures using not only pharmacological but also genetic autophagy inhibition of the ibotenate-induced autophagy. Strategies inhibiting autophagy could then represent a promising neuroprotective approach in the context of severe preterm brain injuries.
PDF
