Current Evidence on Cell Death in Preterm Brain Injury in Human and Preclinical Models

Abstract

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.

Keywords: apoptosis; autophagic cell death; autophagy; necrosis; neonatal; neuroprotection; periventricular leucomalacia.

FIGURE 1

Morphological characteristics and most currently used markers of the three main types of cell death. Analysis of the dying cell ultrastructure is the most reliable method to identify the cell death type. The hallmark of apoptotic cell death (or type I) is a modification of the nuclear structure with presence of chromatin condensation and nuclear fragmentation. The cytoplasm shrinks and plasma membrane deforms until it forms apoptotic bodies that will be removed by phagocytic cells. Molecular markers of apoptosis are caspases (mainly caspase-3) and sometimes cytochrome c (Cyc) mitochondrial release in the cytosol. As a marker of caspase-independent apoptosis, nuclear translocation of AIF (Apoptosis-Inducing Factor) is mainly used. Pyknosis (condensation) of the nucleus shown by H&E (hematoxylin/eosin) or Hoechst stainings is also a main feature of apoptosis. TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) is a staining revealing DNA fragmentation used as a marker of apoptosis with caution if not complemented with a cleaved-CASP3 immunostaining. Autophagic cell death (or type II) is mainly characterized by the presence of numerous autophagosomes (multimembrane vesicles containing intact cytoplasmic material such organelles) and autolysosomes (electron dense structure due to material at different stage of degradation). To conclude on autophagy flux with biochemical markers, it is necessary to study both autophagosome formation and autolysosome degradation. In autophagic cell death, both processes have to be enhanced. Autophagosome presence is reflected by the number of LC3-positive vesicles or by LC3-II level of expression on immunoblot. When autophagic degradation activity is enhanced, vesicles positive for lysosomal markers [such as LAMP1 (lysosomal-associated membrane protein 1) or cathepsins] are increased in number and size since autolysosomes are larger than primary lysosomes. The decrease in p62/SQSTM1 (Sequestosome-1), an autophagosome cargo protein degraded selectively by autophagy, reveals enhanced autophagy. In some cases, the level of autophagy-related (ATG) proteins such as BECN1 (BECLIN1/ATG6), ATG7, ATG5/ATG12, or ATG14 is increased. In necrotic cell death (or type III), ultrastructural changes are mainly cytoplasmic with organelles and cell swelling and presence of empty vacuoles. Perinuclear space is also dilated. Plasma membrane integrity can be finally lost, inducing a strong inflammatory response. Very few tools are available to identify necrotic cell death and mainly indirect markers are used such as strong inflammation response or Ca²⁺-dependent activation of calpains [mainly suggested by the production of a 145- to 150-kDa calpain-dependent cleavage of spectrin (fodrin)]. Necroptosis type of necrosis could be investigated by RIP3/RIPK3 (Receptor-interacting serine/threonine-protein kinase) or MLKL (mixed lineage kinase domain-like) expression and activation.

FIGURE 2

Autophagy and autophagic cell death. (A) Schematic illustration of (macro)autophagy degradation and recycling process showing the following: (1) isolation membrane formation from various possible intracellular membrane sources; (2) elongation and incurvation of the preautophagosome around the cytoplasmic material that has to be degraded (proteins, organelles); (3) closure end-to-end of the preautophagosome forming the autophagosome, a double-membrane vesicle containing undigested materials; (4) fusion of autophagosome with endosomes; (5) fusion of this amphisome with a lysosome to form a mature autophagosome able to degrade its content (autolysosome) thanks to lysosomal hydrolases. LC3 = Microtubule-associated protein 1A/1B-light chain 3 (and other family members). LC3 is the main marker of autophagosome. TGN = trans-Golgi network, ER = endoplasmic reticulum. (B) Electron micrographs showing CA3 neurons presenting features of autophagic cell death 24 h after perinatal hypoxia–ischemia (HI) in P7 rats (unilateral common carotid artery occlusion followed by 2 h of hypoxia at 8% of oxygen). Dying neurons displayed both numerous multimembrane vacuoles loaded with cytoplasmic material (autophagosomes, arrow) and electron dense vesicles containing digested material (autolysosomes, arrowhead). Note that nuclei (N) did not show chromatin condensation. Electron micrographs of CA3 neurons from sham-operated rat brain present healthy neurons. This study was carried out in accordance with the Swiss National Institutional Guidelines for Animal Experimentation. All experiments and methods were approved by the Veterinary Office of the Canton de Vaud. (C) Scheme illustrating the dual role of autophagy in neurons. At basal level, autophagy maintains cellular homeostasis. A reduction or impairment of autophagy can lead to neurodegeneration (such as for some proteinopathies). Autophagy can be activated, above its basal level, as a survival response to maintain energy level during a period of starvation, for example, or to eliminate invading bacteria or virus. However, in other stress conditions such as excitotoxicity, enhanced autophagy could lead to cell death as a mechanism of cell death by itself, independently of apoptosis or necrosis (autophagic cell death or type II), or as a mediator of another type of cell death, mainly apoptosis (autophagy-mediated cell death).