Perspective Chapter: Role of Caspase-3 as Neonatal Hypoxic Ischemic Encephalopathy Biomarker

Abstract

Neonatal hypoxic-ischemic encephalopathy (HIE) is a severe form of neonatal brain damage caused by decreased cerebral blood flow and hypoxia and can cause various serious irreversible neurological sequelae. An early diagnosis of HIE is essential for subsequent treatment and prognosis. Caspase, a protease enzyme that has an essential role in the apoptosis of programmed cell death, is one of the promising biomarkers for diagnosing HIE. Caspase-3 is recognized for its activated proteolytic apoptosis role in cells responding to specific extrinsic or intrinsic inducers of this mode of cell death. Caspase-3 is activated within 1 to 3 hours after neonatal hypoxic-ischemia and is a principal executioner of apoptosis. The role of caspase-3 in apoptosis, pyroptosis, necroptosis, and autophagy might be more profound than its role in cell death. Such functions of caspase-3 require further exploration, however, as there are still many possibilities for its roles in clinical diagnosis and treatment.

1. Introduction

Neonatal hypoxic-ischemic encephalopathy (HIE) is a severe form of neonatal brain damage caused by decreased cerebral blood flow and hypoxia, leading to asphyxia as a clinical sign. HIE is a diffuse disruption of brain function and structure; the development of hypoxic-ischemia can cause a variety of serious irreversible neurological sequelae, including epilepsy, delayed growth and development, cognitive impairment, intelligence disturbance, cerebral palsy, and even neonatal death. Hypoxic-ischemic insults may lead to necrosis or apoptosis of brain cells, depending on the severity of the insults and the cell’s maturity [1].

Perinatal asphyxia occurs two times per 1000 live births in wealthy nations, although it can happen up to 10 times more frequently in underdeveloped nations. Up to 25% of afflicted newborns survive with lifelong neurologic abnormalities, and 15–20% of affected infants die during the neonatal period [2, 3]. An early diagnosis of HIE is essential for subsequent treatment and prognosis. There are several known biomarkers studied in relation to HIE, but their usefulness is limited (S100 protein, interleukin-6, and glial fibrillary acidic protein [GFAP]) [4] or does not correspond to prognosis (blood pH, serum lactate, aspartate aminotransferase [AST], alanine transaminase [ALT], prothrombin time [PT], activated thromboplastin time [APTT], creatinine, urea, troponin T, and fibrinogen) [5]. Caspase is one of the promising newer biomarkers for diagnosing HIE. A family of protease enzymes known as caspase plays a crucial part in apoptosis, or “programmed cell death.” Caspase is short for cysteine-dependent aspartate-directed protease. There are 12 caspases in humans, and these are categorized into three subtypes based on their function: Initiator caspases (caspase 2, 8, 9, and 10) signal apoptosis; executioner caspase (caspase 3, 6, and 7) leads to apoptosis; and inflammatory caspases (caspase 1, 4, 5, 11, and 12) organize inflammatory cytokine signaling and pyroptosis [6].

Caspase-3 is one of the most important components in programmed cell death as it catalyzes the specific cleavage of many essential cellular proteins. By understanding the role and mechanism of caspase-3 in neonates with HIE, it might be possible to use caspase-3 as a biomarker to diagnose HIE and/or even prevent and treat HIE promptly.

2. Pathophysiology of hypoxic-ischemic encephalopathy

Intermediate anoxia or acute hypoxia in neonatal encephalopathy leads to decreased cerebral perfusion. A biphasic process of primary and secondary energy failure at the cellular level distinguishes systemic hypoxemia and cerebral hypoperfusion. The initial phase consists of triggering the hypoxic-ischemic interval that leads to primary energy failure (a reduction in high-energy phosphorylated metabolites and intracellular pH). This phase may be severe, resulting in permanent brain injury or subacute brain injury if the case is responsive to resuscitation [7].

The phases in hypoxic-ischemic encephalopathy consist of primary energy failure, latent phase, secondary energy failure, and tertiary phase. First, an immediate primary neuronal injury occurs due to the brain’s interruption of oxygen and glucose. The associated decrease in adenosine triphosphate (ATP) production results in the failure of the ATP-dependent Na-K pump. Sodium enters cells followed by water causing cell edema, widespread depolarization, and cell death. Cell death and lysis cause the release of glutamate, an excitatory amino acid, which causes an increase in intracellular calcium influx and further cell death [7, 8].

The acute phase, also known as primary energy failure, is characterized by anaerobic metabolism, oxidative stress, neuronal cell death, and excitotoxicity. In the process of excitotoxicity, an excessive amount of the excitatory amino acid, glutamate, leads to overstimulation of the 2-(aminomethyl)phenylacetic acid (AMPA), kainite (KA), and N-methyl-D-aspartate (NMDA) receptors. Overstimulation of AMPA and KA causes an influx of sodium (Na+) and chloride (Cl−), which leads to an increased cellular osmolality. Overstimulation of NMDA triggers the influx of calcium (Ca2+), which leads to apoptosis and necrosis. The mitogen-activated protein kinase (MAPK) signaling pathway is overactivated after the hypoxic-ischemic event, leading to further neuronal apoptosis. Prostanoids also play a role in the hypoxic-ischemic event: Stimulating prostanoid receptors may be neuroprotective and prevent cerebellar neuronal injury. A partial recovery with reperfusion happens 30 to 60 minutes after the hypoxic-ischemic event in a penumbra of the brain, depending on the length of reduced perfusion and the presence or absence of medical intervention [8, 9].

The severity of the original energy failure influences how badly the secondary energy failure phase will be. Necrosis may result in neuronal cell death if the hypoxic-ischemic insult is severe. There is a brief time of recuperation when blood flow has been restored. Normal brain metabolism is present throughout this brief time of recuperation, known as the latent phase. The hypoxic-ischemic insult’s intensity and magnitude are hypothesized to have an impact on the latent period. The latent period shortens with increasing injury severity. It is currently uncertain exactly when the latent period, the secondary energy failure phase, and the primary energy failure phase start and terminate. The ideal time for therapeutic treatments is thought to be during the latent phase. [9] The latent phase may last 1 to 6 hours. This latent phase is characterized by neuroinflammation and the continuation of an activated apoptotic cascade. In neonates with moderate to severe HIE, the latent phase is followed by the secondary phase, also known as secondary energy failure [8].

Neuronal cell death may be immediate or delayed and results from neuronal apoptosis or necrosis. Activation of proteases and endonucleases, neuronal apoptosis, microglial activation, reduction of growth factors and protein synthesis, and accumulation of excitatory neurotransmitters happened within approximately 6 to 15 hours. This secondary phase also caused cytotoxic edema, excitotoxicity, and cerebral hyperperfusion. The tertiary phase occurs in the weeks or months following primary energy failure. It involves remodeling the injured brain, astrogliosis, and late cell death [8, 9, 10, 11].

The most important molecular trigger of apoptosis is an influx of Ca2+ and the generation of reactive oxygen species (ROS). These membrane perturbations involve the release of ceramide from sphingomyelin, translocation of the mitochondrion of proapoptotic member Bc1-2 family formation of the mitochondrial permeability pore, and release from the mitochondrion of cytochrome c and apoptosis-inducing factor and of caspases, especially caspase-3 by cytochrome c [8, 9, 10].

3. Role of Caspase-3 in programmed cell death

Caspase-3 is a lysosomal enzyme involved in the apoptotic pathway. It is a caspase protein that is encoded by the CASP3 gene. Caspase-3 is recognized for its activated proteolytic apoptosis role in cells responding to specific extrinsic or intrinsic inducers of this mode of cell death [12].

All caspases are initially created as dormant zymogens (known as procaspases), which are later transformed to activation by a variety of distinct internal and/or external cues. Procaspase-3 is made up of two subunits, one big and one small, known as p20 and p10, respectively, as well as an N-terminal prodomain. These two components work together to form the mature protease’s catalytically active pocket [13].

Caspase-3 is synthesized as an inactive 32 kDa proenzyme and is processed during apoptosis generating two subunits of 17 and 12 kDa. Caspase-9 cleaves and activates caspase-3, which is present in the cell as an inactive proenzyme. Caspase-3 is activated within 1 to 3 hours after neonatal hypoxia-ischemia and is a principal executioner of apoptosis [10].

The particular cleavage of several cellular proteins is catalyzed by caspase-3, a death protease that is regularly activated. Whether reliant or independent of mitochondrial cytochrome c release and caspase-9 action, pathways to caspase-3 activation have been discovered. Caspase-3 is essential for apoptotic chromatin condensation and DNA fragmentation in all cell types, as well as for other classic apoptotic markers, throughout normal brain development [14].

4. Caspase-dependent mechanisms of apoptotic cell death

Cell death may be achieved by various mechanisms. During development and throughout life in cell renewal tissues, cells are continuously eliminated by mechanisms that induce apoptosis. Caspases play a crucial role in programmed cell death (apoptosis). Two main pathways have evolved for activating the caspase cascade: The mitochondrial pathway (intrinsic pathway) and the death receptor pathway (extrinsic pathway) [13].

A hypoxic event initiates the intrinsic pathway. The permeabilization of the mitochondrial outer membrane (MOMP) is one of the important stages in this process. The Bcl-2 family members provide the main function in MOMP. These proteins are classified as proapoptotic (Bax, Bak, etc.) and antiapoptotic (Bcl-2, Bcl-XL, etc.) proteins based on whether they include one or more Bcl-2 homolog (BH) domains. Subgroups of proapoptotic members have also been established: The BH3-only proteins and the BH1, BH2, and BH3-containing proteins. Bax and Bak are prone to conformational changes and oligomerization as a result of apoptotic stimuli, and they produce MOMP by disrupting the lipid bilayer, either creating pores or interacting with channels. Bax and Bak are counteracted by Bcl-2 antiapoptotic members, which are present in the outer membrane. In this regulation, BH3-only proteins have an essential role. A consequence of such proapoptotic cellular stressors is the permeabilization of the mitochondrial outer membrane and the release of apoptogenic factors such as cytochrome c into the cytoplasm. Cytochrome c molecules bind to an adaptor protein, apoptotic protease activating factor 1 (APAF-1), which recruits initiator caspase-9. This leads to the formation of the APAF1-containing macromolecular complex called the apoptosome [15, 16, 17].

Mature caspase-9 remains bound to the apoptosome, recruiting and activating executioner caspase-3 and caspase-7. This complex, in turn, binds and activates procaspase 9. Inhibitors of apoptosis (IAPs), of which x-linked IAP (XIAP) is the sole direct caspase-3 inhibitor, block the activation of caspase-3 and -7, while Smac/Diablo and Omi/HtrA2, which are released from the intermembrane space when mitochondria are injured, can also block this process [15, 17].

The extrinsic pathway includes cell surface receptor signaling. Death receptor-like Fas or Fas ligand (FasL) binding leads to receptor trimerization and recruitment of specific adaptor proteins. The Fas receptor contains a death domain (DD) in its cytoplasmic region that interacts with the adaptor protein. Extracellularly-mediated ligand binding to death receptors causes caspase-8 to bind to the Fas-associated death domain (FADD) adaptor protein, thereby forming a death-inducing signaling complex (DISC). FADD contains a death effector domain (DED), which recruits the DED-containing procaspase-8 into the DISC [16, 17].

Procaspase-8 is proteolytically activated to the enzymatically active caspase-8, activating downstream effector caspases. The oligomerization and activation of DISC are facilitated by the recruitment of caspase-8, which self-cleaves. The ultimate steps of apoptotic cell death are then brought about by the activation of downstream effector caspases, which is caused by cleaved caspase-8 [15].

The death receptor and mitochondrial-associated death pathways may interact in some cell types. In this instance, the proapoptotic Bcl-2 family member Bid is cleaved by caspase-8, activating the mitochondrial pathway. Truncated Bid (t-Bid) translocate to the mitochondria after being cleaved, where they cause the release of cytochrome c, an important step in the mitochondrial process. [16, 17].

Together with other effector caspases (including caspase-7 and -6), the extrinsic and intrinsic pathways come together at caspase-3 to plan the destruction of certain cell structures through the cleavage of a particular substrate. These caspase-mediated cleavages produce phenotypic changes in the apoptotic cell [15].

5. Caspase-independent mechanisms of apoptotic cell death

Caspase-independent cell death (CICD) ensues when a signal generally induces apoptosis and fails to activate caspases. Activation of caspases can occur after the ligation of death receptors or after the release of proapoptotic factors from mitochondria. Bcl-2 family proteins control the latter pathway. The APAF-1 binds to the caspase recruitment domain (CARD) in this pathway, triggering the activation of caspase-9 as the initiator. The proapoptotic factor cytochrome c causes caspase-9 and Apaf-1 to associate, leading to the development of apoptosomes. This component is found in the intermembrane gap of mitochondria, which also contributes to electron transport during respiration [13, 18].

Cytochrome c is released from the intermembrane gap during apoptosis as a result of increased permeability of the mitochondrial outer membrane. This process is triggered and controlled by pro- and antiapoptotic Bcl-2 family proteins [13, 18].

In cells lacking apoptosome-mediated caspase activation due to a lack of caspase-3, caspase-9, or Apaf-1, excessive Bax and Bak stimulation can cause mitochondrial dysfunction-triggered death. Bax and Bak must both be activated for this mitochondrial step because neither can start the intrinsic apoptotic pathway by itself [17].

Mitochondria, however, can release multiple factors that may trigger caspase-independent cell death. Moreover, cell death may be caused by altered mitochondrial energetics directly due to the loss of cytochrome c.

Numerous mitochondrial intermembrane space proteins are produced during MOMP in the absence of caspase activation, precipitating loss of mitochondrial function and potentially causing cell death. The simultaneous activation of mitochondrial fusion and inactivation of mitochondrial fission causes a disruption in mitochondrial morphology. CICD is influenced by these occurrences. The mechanism by which MOMP can follow CICD is through either a general decline in mitochondrial function or the release of mitochondrial proteins that can induce CICD [13, 18].

6. Necrotic cell death mediated by caspases

The following is the necrosis mechanism mediated by inflammatory caspase. The proinflammatory subfamily of caspases, counting caspase-1, caspase-4, caspase-5, and caspase-11, is known to intercede in a type of necrotic cell passing named pyroptosis (Greek roots pyro, connecting with fire or fever), which is portrayed by cell enlarging, lysis, and arrival of proinflammatory cytokines and intracellular substance [19].

When stimulated by pathogens such as bacteria, viruses, or their byproducts such as lipopolysaccharide (LPS) and viral DNA, phagocytes such as macrophages, monocytes, and dendritic cells (DCs), as well as other cell types (such as T cells), undergo pyroptosis. Like apoptosis, the critical execution mechanism of pyroptosis involves cleavage events mediated by caspases. The ability of inflammatory caspases to promote cell lysis involves gasdermin D (GSDMD) cleavage to promote membrane formation. Inflammasomes may activate inflammatory caspases mediating pyroptosis. The inflammasome pathways include the canonical inflammasomes that mediate the activation of caspase-1 and the noncanonical inflammasome that promotes the activation of caspase-4, 5, and [13, 19, 20].

Human caspase-1, caspase-4, and caspase-5 are identified as gene clusters on chromosome 11. Caspase-1, the enzyme directly involved in the processing of pro-IL-1β, is subject to regulation by caspase-4, -5, and -11 [18]. The activation of pyroptosis is proinflammatory as opposed to apoptosis, which largely has anti-inflammatory effects. This is due to the processing and release of mature IL-1 and IL-18, which have strong proinflammatory activity in promoting vasodilation and extravasation of immune cells, the generation of IL-17-producing helper T cells-mediated (Th17) response, and the production of interferon- (IFN-) by NK and Th1 cells [19, 21]. Additionally, this is due to the rapid loss of cell membrane integrity and release of cytosolic contents.

Caspases also play a role in the program of cell death through necrosis. For this necrotic cell death, however, caspase-3 plays a direct role. Several studies have shown a relationship between caspase-3 and the process of necrosis. It is known that LPS is a highly induced proinflammatory caspase for mediating sepsis but also capable of mediating the cleavage of downstream caspases such as caspase-3/-7. It was likewise found that caspase-3 severs the GSDMD-related protein DFNA5 after Asp270 to produce a necrotic DFNA5-N section that causes the plasma layer to instigate optional rot/pyroptosis [22, 23].

The role of caspase-3 might be more profound than explained above; we need to explore more of its role in apoptosis, pyroptosis, necroptosis, and autophagy. There is emerging evidence that caspase-3 also has a pivotal role in regulating the growth and homeostatic maintenance of both normal and malignant cells and tissues in multicellular organisms. There are still many vast possibilities for the role of caspase-3 and its use in the clinical identification and treatment.

7. Conclusions

Caspase-3 is one of the most essential components in programmed cell death due to its catalyzing the specific cleavage of many essential cellular proteins. Caspase-3 is activated within 1 to 3 hours after neonatal hypoxic-ischemia and is a principal executioner of apoptosis. Pathways to caspase-3 activation have been identified whether dependent or independent of mitochondrial cytochrome c release and caspase-9 function.

Conflict of interest

The authors declare no conflict of interest.

List of abbreviations

AMPA

2-(aminomethyl)phenylacetic acid

APAF-1

apoptotic protease activating factor 1

ALT

alanine transaminase

APTT

activated partial thromboplastin time

AST

aspartate aminotransferase

ATP

adenosine triphosphate

BH

Bcl-2 homolog

DD

death domain

CARD

caspase recruitment domain

CICD

caspase-independent cell death

DC

dendritic cells

DED

death effector domain

DISC

death-inducing signaling complex

FADD

Fas-associated death domain

GFAP

glial fibrillary acidic protein

GSDMD

gasdermin D

HIE

hypoxic-ischemic encephalopathy

IAP

inhibitors of apoptosis

IFN

interferon

KA

kainite

LPS

lipopolysaccharide

MAPK

mitogen-activated protein kinase

MOMP

mitochondrial outer membrane

NMDA

N-methyl-D-aspartate

PT

prothrombin time

t-Bid

truncated Bid

ROS

reactive oxygen species

XIAP

x-linked inhibitors of apoptosis