Mechanisms of Cell Death in the Developing Brain

Introduction

When the brain is exposed to stress, a number of adaptive responses can act to reestablish homeostasis. However, when the stress is severe and/or the endogenous protective processes are not sufficiently effective, the cell will die. ^, Cell death can be initiated by numerous organelles (e.g., nucleus, mitochondrion, endoplasmic reticulum/lysosome, cytoskeleton, plasma membrane) and mitochondria, in particular, play a key role in the initiation and execution of cell death mechanisms in the immature brain. ^, Once triggered, cell death can proceed via a variety of routes, including the well-characterized apoptosis pathway, as well as by lesser-known necroptosis, ferroptosis, parthanatos, and autophagy-dependent cell death mechanisms ( Fig. 125.1 ). Which cell death mechanism predominates will depend on the metabolic state, severity and type of insult, cell type, developmental age, and other factors. ^, The traditional classification of cell death by morphology is no longer used, as mixed morphologic phenotypes are often detected in many pathologic situations. ^, Classification now favors the use of molecular markers indicative of particular cell death pathways. Indeed, for effective neuroprotective intervention, understanding the biochemical route(s) to cell death is critical, considering that when one route is inhibited, cell death may occur via an alternative mechanism. ^, Long-term functional cell recovery following genetic alteration and/or pharmacologic intervention often yields significant information concerning the essential components in a specific route of cell death and any alternative routes triggered. More recently, unbiased proteomics approaches have provided a more comprehensive identification of the major players in a variety of cell death paradigms. ^{, ,}

Cell death can be classified into accidental and regulated (see Fig. 125.1 ). Accidental cell death is evoked by severe insults (such as severe trauma, core of an ischemic infarct, high temperature), which cause immediate cellular demise that does not involve a specific molecular mechanism and cannot be prevented or modulated. However, cellular contents released following accidental cell death act as damage-associated molecular patterns (DAMPs) often having direct toxic effects on surrounding cells that survived the initial insult. Exposure to DAMPs may extend the primary injury, as they have immunogenic properties, contributing to an inflammatory response that may cause injury and aggravate the situation further. ^, Various interventions that attenuate DAMP-induced cellular actions can provide protective effects. So even if accidental cell death cannot be targeted directly, its consequences can be intercepted and bystander injury prevented to some extent. For example, therapeutic hypothermia is used in mitigation of the effects of hypoxic-ischemic injury. In contrast, regulated cell death involves the coordinated molecular machinery of the cell (see Fig. 125.1 ), and its course can be modulated by pharmacologic and genetic means. ^, Regulated cell death usually occurs with a delay, in situations when endogenous protective mechanisms fail to restore cellular homeostasis.

In the developing brain, cell damage can be induced by a variety of insults, such as hypoxia, hypoxia-ischemia (HI), trauma, and infections. While definitive studies using human neonatal brain tissues are lacking, substantial knowledge of the mechanisms of cell death in the immature central nervous system has emerged from in vitro studies in cell cultures (exposed to toxins or oxygen/glucose deprivation) and in vivo, in models of HI in rodents and to some extent larger animals (e.g., piglets, rabbits, or fetal sheep). Exposure to HI results in an initial depletion of high-energy phosphates, in particular ATP and phosphocreatine. The levels of these phosphates return transiently to the baseline, but this is followed by a second, more prolonged depletion of cellular energy reserves accompanied by progression of brain injury (see Chapter 167 ). ^, These disturbances in energy metabolism trigger a number of pathophysiologic responses that ultimately lead to cell death, with mechanisms including initial necrosis, and subsequently apoptosis, , necroptosis or autophagy-dependent cell death.

This chapter will describe the mechanisms underlying the most common forms of regulated cell death and then summarize the evidence for their involvement in cell death in the immature brain exposed to hypoxic-ischemic insult.

The Apoptotic Cell Death Machinery

The apoptosis pathway triggered depends on whether the stimulus is intracellular (intrinsic, mitochondrial) or extracellular (extrinsic), although both converge at the activation of caspases-3 and -7 ( Fig. 125.2 ). The intrinsic pathway relies on mitochondrial outer membrane permeabilization (MOMP) resulting in the release of a number of proapoptotic proteins into the cytosol. This permeabilization is tightly controlled by the pro- and antiapoptotic members of the B cell lymphoma 2 (BCL2) family. MOMP is directly executed by BCL2-associated X protein (BAX) and BCL2-antagonist/killer 1 (BAK1) that oligomerize to form outer membrane pores (see Fig. 125.2 ). The opening of the BAX/BAK1 pore is regulated by the balanced action of antiapoptotic BCL2 family proteins such as BCL2 itself, BCL2-like 1 (BCL-XL), and myeloid cell leukemia 1, and proapoptotic “sensitizers” such as BCL2 binding component 3 (PUMA), BCL2-like 11 (BIM), and BH3-interacting domain death agonist (BID), which can initiate the pathway. Initiation of MOMP can also be controlled by p53, c-Jun N-terminal kinase (JNK), and caspase-2. Once MOMP is initiated, leakage of proapoptotic proteins from the mitochondrial intermembrane space occurs, most notably cytochrome c (Cyt c ). Cyt c forms an apoptosome complex with deoxy-ATP, and apoptotic peptidase-activating factor 1 (APAF-1), which recruits and activates caspase-9, leading to the downstream activation of the executioner caspase-3. ^, Additional proapoptotic proteins released from the mitochondrion include Smac/DIABLO and Omi/HtrA2, proteases that cleave X-linked inhibitor of apoptosis (XIAP), preventing XIAP-mediated inhibition of caspase activation. Finally, apoptosis-inducing factor (AIF) is also released from the mitochondrion, translocating to the nucleus where it facilitates chromatinolysis and cell death in a caspase-independent manner. MOMP is not only very rapid but also extensive; once initiated in a limited number of mitochondria, MOMP swiftly proceeds through all mitochondria within the cell.

In the extrinsic pathway, binding of extracellular ligands to a death receptor leads to activation of caspase-8 and cleavage of either BID (to crossover with the intrinsic pathway) or directly to caspase-3. Over 20 ligand-receptor pairings are now included in the death receptor ligand tumor necrosis factor (TNF) superfamily. These TNF-receptor (TNFR) and TNFR-like molecules are similar in structure to TNF and function as trimers (both ligands and receptors). Because of the similarity of their structure, multiple ligands are able to bind and induce signaling through one receptor, or a single ligand is able to bind multiple receptors. Receptors containing the so-called death domain in their intracellular domain (e.g., TNFR1, DR4, DR5, Fas) are able to trigger apoptosis when activated by the binding of the corresponding ligand (e.g., TNF-α, TNF-related apoptosis inducing ligand [TRAIL], FasL). This extrinsic pathway of apoptosis continues with the activation of a death-inducing signaling complex (DISC) adjacent to the death domain of the receptor. DISC comprises Fas-associated death domain protein (FADD) and procaspase-8; binding results in caspase-8 homodimerization and activation by proteolytic cleavage (see Fig. 125.2 ). Activated caspase-8 either directly activates caspase-3 or mediates cleavage of BID to truncated BID (tBID), which integrates different death pathways at the mitochondria. Truncated BID translocates to mitochondria, where it interacts with other proapoptotic proteins and triggers the release of apoptogenic factors, leading to caspase-dependent and caspase-independent cell death. Death receptors can also trigger necroptosis, particularly under conditions when caspase-8 is inactive (see the section “Necrosis and Necroptosis”).