Caspase-3 Inhibition toward Perinatal Protection of the Developing Brain from Environmental Stress

Throughout our lives, we are exposed to a variety of hazards, such as environmental pollutants and chemical substances that affect our health, and viruses and bacteria that cause infectious diseases. These external factors that are undesirable to an organism are called environmental stress. During the perinatal period, when neural networks are drastically reorganized and refined, the tolerance of the developing brain to various environmental stresses is lower than in adulthood. Thus, exposure to environmental stress during this vulnerable period is strongly associated with cognitive and behavioral deficits in later life. Recent studies have uncovered various mechanisms underlying the adverse impacts of environmental stress during the perinatal period on brain development. In this mini-review, we will present the findings from these studies, focusing on caspase-mediated apoptotic and nonapoptotic effects of environmental stress, and discuss several compounds that mitigate these caspase-mediated effects as examples of potential therapeutic approaches.


Introduction
Undisturbed prenatal brain development is key to a healthy future, as evidenced by what happens when there are abnormal prenatal conditions. Acute or repeated prenatal exposure to harmful environmental stress (ES) can have a wide range of unpredictable negative effects on brain development and function [1]. For example, prenatal exposure to harmful chemicals such as ethanol, methylmercury, and pentylenetetrazol activates heat shock factor 1 (HSF1), a major stress response transcription factor, in the embryonic mouse and human brain, but the level of activation is unpredictable from exposure dose This article is licensed under the Creative Commons Attribution 4.0 International License (CC BY) (http://www.karger.com/Services/ OpenAccessLicense). Usage, derivative works and distribution are permitted provided that proper credit is given to the author and the original publisher.
[2]. Although such HSF1 activation in response to prenatal exposure to ES generally plays critical roles in cell protection [3], its overactivation in a subset of cells can cause brain malformation [4]. A recent study has also shown that prenatal exposure to ES has long-term impacts on gene expression in those cells in which HSF1 is once overactivated; among the genes altered in these cells in the brain of mice prenatally exposed to ethanol is Kcnn2, which encodes small conductance calcium-activated potassium channel protein 2, whose abnormal increase in the motor cortex is directly involved in motor learning deficits. Indeed, pharmacological blockade of KCNN2 in these mice improves their learning deficits [5]. As another example, a single maternal injection of interleukin-6, as a mouse model of maternal immune activation, during neurogenesis results in deficits in cognitive and exploratory behaviors in the offspring [6]. Treating gastrointestinal barrier defects in these offspring with probiotics improves their behavioral abnormalities, although the mechanism underlying this improvement remains to be explored [7,8]. Thus, much research has progressed on the impacts of prenatal exposure to ES on brain development and the potential treatment and intervention for the deficits caused by such impacts.
The significance of the postnatal environment for brain development also cannot be overstated. It is widely recognized that children exposed to various forms of ES in the first year of life are associated with alterations in brain development that correlate with neural, behavioral, and psychological health problems [9]. For example, repeated exposure to anesthesia with ketamine-xylazine in mice during postnatal day (P)7-P11 or P14-P18 impairs motor learning and learning-dependent dendritic spine plasticity later in life through mechanisms independent of apoptotic cell death [10,11]. In postnatal neural development, different brain areas have their own critical periods, or periods when neuroplasticity is particularly heightened [12]. During the critical period, neural circuits are fine-tuned using sensory inputs from the animal's environment, eliminating redundant synapses and strengthening useful connections. Sensitivity to ES also becomes significantly higher during this period. After neural circuits are consolidated, the critical period ends. Therefore, the critical period is an important time window to protect the developing brain from various ES, and new treatments or protocols could improve the quality of life for patients who experience the negative effects of disturbances during this critical period. Below, we will briefly introduce the apoptotic and nonapoptotic roles of caspase activation in normal brain development and in exposure to ES and summarize the effects of compounds that directly or indirectly suppress caspase activation. We will then discuss the potential clinical applications of these compounds.

Apoptotic and Nonapoptotic Impacts of Caspase Activation by Exposure to ES during the Neural Vulnerable Period
The caspase cysteine protease family is the central mediator of programmed cell death via apoptosis [13]. Using the activation of caspase-3, a major effector of the caspase pathway, as a marker, it has been demonstrated that neurons in the cerebral cortex are particularly sensitive to exposure to various ES, such as ethanol [14,15] and general anesthetics during a specific period (P0-P14 in mice: the neural vulnerable period) [16][17][18]. Activation of caspase-3 by such exposure is observed in neurons throughout many cortical regions in mice at P7, which is roughly equivalent to the third trimester of pregnancy in humans [14,15], consistent with the concept of elevated vulnerability of developing brain during the brain growth spurt period [19].
In addition to their best-known roles in regulation of apoptotic cell death, activated caspases also contribute to nonapoptotic events, including pruning of axons and dendrites through cleavage of proteins like β-actin and α-tubulin [13,20] both in normal brain development and in exposure to ES [21,22]. While spatiotemporally controlled caspase activation plays essential roles in normal brain development [23][24][25], excessive caspase activation due to ES exposure causes devastating effects [13,22]. Thus, the inhibition of caspase activation during the neural vulnerable period, whether in apoptotic or nonapoptotic processes, is one of the major targets to maintain the normal number, morphology, and function of neurons in the developing brain that has been exposed to ES (Fig. 1). It should be noted, however, that inhibition of normal caspase function can also have detrimental effects.

Compounds That Suppress Caspase-3 Activation Caused by Exposure to ES during the Neural Vulnerable Period
In this section, we introduce several compounds that mitigate caspase activation-mediated abnormalities in the developing brain exposed to ES during the prenatal or DOI: 10.1159/000529125 postnatal period, to explore potential approaches toward developing early-life pharmacological interventions. These compounds are summarized in Table 1.

Direct Caspase Inhibitors
Quinoline-Val-Asp-Difluorophenoxymethylketone Q-VD-OPh (quinoline-Val-Asp-difluorophenoxymethylketone) is a third-generation broad-spectrum pancaspase inhibitor. This compound is far more effective than the widely used pan-caspase inhibitor z-VAD-fmk and Boc-D-fmk. The structural design of Q-VD-OPh differs significantly from that of z-VAD-fmk and Boc-Dfmk; the carbobenzoxy-blocking group (z) was replaced with a quinoline derivative (Q), the tripeptide sequence was changed from VAD (valine-alanine-aspartic acid) to VD (valine-aspartic acid), and the fluoromethyl ketone (fmk), which is highly toxic after conversion into fluoroacetate, was replaced by the nontoxic 2,6-difluorophenoxy (OPh) group. Many studies have shown the effectiveness of Q-VD-OPh to alleviate the damage in the developing brain caused by various physical and chemical ES. For example, Q-VD-OPh was given to P7 rats who had unilateral focal hypoxia-ischemia (HI) followed by reperfusion, which causes brain injury with apoptosis-like characteristics [26]. Time course and pattern of caspase-3 activation in this injury differs between genders due possibly to differential mitochondrial response [26]. After 48 h of treatment with Q-VD-OPh, significant neuroprotection was observed in females. This effect persisted and reduced neurological dysfunction after 21 days. There was no significant effect in males [26]. The effectiveness of delayed administration Q-VD-OPh was also investigated; it reduced caspase-3 activity and tissue loss after unilateral HI induction of stroke in P9 male mice when administered acutely at 12 and 36 h after HI [27].
Ac-AAVALLPAVLLALLAPDEVD-CHO Ac-AAVALLPAVLLALLAPDEVD-CHO (Ac-A∼ PDEVD-CHO) is a cell permeable caspase-3 inhibitor (also inhibits caspase-6, -7, -8, and -10). Caspase-3 enzymatic activity increased in the frontal cortex, striatum, and hippocampus in a dose-dependent manner when P7 rats were given phencyclidine (PCP), an aryl cyclohexamine that can mimic certain symptoms of schizophrenia Caspase-mediated apoptotic and nonapoptotic effects of ES. By exposure to various ES, such as hypoxia, ethanol, and general anesthetics, during the neural vulnerable period, cleavage (activation) of caspase-3 is abnormally increased in the brain. Cleaved caspase-3 leads to neuronal death and pruning of axons and dendrites through the cleavage of cytoskeletal proteins like β-actin and α-tubulin, thereby leading to cognitive and behavioral deficits in later life. Several compounds that inhibit the activation of caspase-3 may mitigate these excess caspase-mediated apoptotic or nonapoptotic effects and restore normal cognition and behavior as an example of a potential therapeutic approach. due to the use-dependent blockade of the N-methyl D aspartate receptor antagonists [28]. At 3 μM, Ac-A∼PDEVD-CHO completely inhibited PCP-induced caspase-3 activation in brain slices, and the pretreatment significantly prevented PCP-induced neuronal deaths in a concentration-dependent manner. According to terminal deoxynucleotidyl transferase dUTP nick end labeling staining, this caspase inhibitor prevented 85 percent of PCP-induced neuronal death in vitro [29].

Indirect Caspase Inhibitors
Osmotin Osmotin, a tobacco protein and adiponectin homolog, exhibits anti-apoptotic effects by reducing the protein level of cleaved caspase-3 through adiponectin receptors [30]. It can enter the cerebrospinal fluid and protect the brain from ischemia [31]. When P7 rats were injected intraperitoneally with 4 g/kg body weight ethanol, osmotin treatment mitigated the ethanol-induced increases in cleaved caspase-3 and reduced the number of annexin-V + apoptotic cells [30]. Fetal rat hippocampal neurons exposed to ethanol plus osmotin for 24 h had fewer degenerating cells than those exposed to ethanol alone [30]. Osmotin increased cell viability by up to 80-90% when compared to the groups exposed to ethanol only. It also protected fetal rat hippocampal neurons against ethanol-induced mitochondrial damage measured via membrane potential; mitochondrial membrane potential was lower when exposed to ethanol and partially rescued under osmotin treatment [30].

Melatonin
Melatonin (N-acetyl-5-methoxytryptamine) is a pineal gland secretory product with neuroprotective properties against ischemia/reperfusion injury [32]. Melatonin's neuroprotective properties have been linked to its powerful endogenous anti-oxidative stress and free radical scavenging properties [33]. To test the effect of melatonin on apoptosis, male and female rat pups were subjected to unilateral HI at P7 [34]. Melatonin was injected intraperitoneally 1 h before HI, and an additional 6 doses were administered at 24-h intervals. The pups that were given melatonin had significantly fewer cortical tissue injuries when compared to their control littermates that received HI but melatonin treatment. Consistently, when compared to the control, melatonin treatment inhibited caspase-3 activation after HI [34].

Nicotinamide
Nicotinamide is a vitamin B3 amide and precursor for the coenzyme beta-nicotinamide adenine dinucleotide, •Inhibition of TUNEL + staining by 87% at 3 μM Wang, 2007 [29] General anesthesia IP at P7 (midazolam 9 mg/kg before 0.75% isoflurane and 75% nitrous oxide) •10-11-fold increase in the number of ROS + pyramidal neurons compared to control •Significant increase in pg 8-isoprostane, a marker of lipid peroxidation •Increased density of abnormal mitochondria which is necessary for cellular function and metabolism [35]. Nicotinamide therapy enhances neuronal survival when exposed to a variety of harmful stimuli, including free radical exposure and oxidative stress [36], and can even mitigate aging-associated physiological decline in mice [37]. To test its effect on brain damage by ethanol exposure, P7 mice were given a single dose of ethanol (5 g/kg), followed by nicotinamide treatment for 8 h. Nicotinamide inhibited caspase-3 activation in the forebrain and prevented behavioral abnormalities, such as impaired performance in the fear conditioning test and increased activity in the open field and plus mazes, observed in mice exposed to ethanol [38].
EUK-134 and R (+) PPX Early exposure to general anesthesia, such as sevoflurane, causes apoptosis in the developing brain as well as long-term cognitive impairment [16]. It also inhibits neuronal mitochondrial development [39], which can be a significant source of reactive oxygen species (ROS). These ROS have the potential to cause lipid peroxidation and membrane damage [40]. EUK-134, a synthetic superoxide dismutase discovered in the 1990s, can scavenge these ROS and degrade both superoxide anions and hydrogen peroxides [41], lowering the risk of harm to the developing brain. Similarly, R (+) pramipexole (PPX), a synthetic aminobenzothiazol derivative, restores mitochondrial integrity. Compared to rats receiving general anesthesia alone, rats treated with EUK-134 or PPX had significantly lower lipid peroxidation, maintained mitochondrial integrity, and experienced significantly less neuronal loss [42].

Lithium
Bipolar disorder, also known as manic depression, is characterized by mood swings between high and low levels, as well as changes in behavior, thinking, energy, and sleep [43]. Lithium has been used for over 6 decades as a treatment for acute manic, mixed, and depressive episodes associated with bipolar disorder, as well as for long-term use in the prevention of such acute episodes [44]. Lithium also reduces suicidal risk to a level comparable to that of the general population [45]. Although the precise mechanism of lithium is still unclear, studies have shown that it directly inhibits glycogen synthase kinase 3, which has various effects on apoptosis, neurotrophic factors, and other biological processes [44]. Lithium administration in mice at P7-P14 after early postnatal exposure to ethanol can reverse density reduction of parvalbumin + interneurons in the barrel cortex, but not in the dentate gyrus [46], and lithium administration immediately following ethanol exposure prevents caspase-3 activation and apoptotic cell death in the barrel cortex. Long-term lithium pretreatment prevents glutamate-induced AKT1 activity loss and speeds its recovery to control levels [47].
Bix 01294 Histone H3 di-methylation (H3K9me2) has been shown to be involved in caspase-3 activation following ethanol exposure [48]. This process is moderated by lysine dimethyl transferase (G9a), and therefore, administration of Bix 01294, a small-molecule inhibitor for G9a before ethanol exposure, prevents ethanol-caused caspase activation [48,49]. Bix 01294 administration in mice at P7 before ethanol exposure also rescued the deficits in long-term potentiation and social recognition memory [50]. These effects of Bix 01294 support the idea that early ethanol exposure has epigenetic effects and inhibition of histone methylation might be beneficial in mitigating these effects.

SR141716A
As with ethanol, cannabis use during neurodevelopment is associated with developmental disorders, and cannabis use causes adverse effects similar to those observed after ethanol exposure, including persistent synaptic and memory deficits [51]. These effects are likely mediated by activation of cannabinoid receptor type 1 [52], an inhibitory G-protein-coupled receptor [53] whose activation is known to inhibit the formation of new synapses by suppressing the formation of cAMP [54]. Endocannabinoids have also been speculated to play a role in ethanol-induced damage in the developing brain through the interaction between ethanol and molecular constituents of the endocannabinoid system [55]. Consistent with these lines of evidence, SR141716A, a potent antagonist of central cannabinoid receptors in the brain [56], significantly reduced ethanol-induced caspase-3 activation in the cortex in a dose-dependent manner. In adult mice exposed to ethanol at P7, this pharmacological inhibition also prevented ethanol-induced impairments in synaptic plasticity and memory [52].

SC79
Cilia, which consist of motile and primary types, are hair-like organelles projecting from the soma of cells including neurons and glia, acting as a kind of cellular antenna [57]. By detecting physical and chemical extracellular cues and transducing those signals into cells [58], primary cilia regulate neuronal cell fate, migration, DOI: 10.1159/000529125 differentiation, and many other cellular behaviors [59]. Contrary to expectation, dorsal telencephalon-specific cilia-deficient mice (Emx1-IRES-Cre; Ift88 floxed mice) exhibit no obvious defects in cortical development and no significant differences in brain size [60,61]. Remarkably, exposure of these mice to ethanol during the neural vulnerable period resulted in significantly higher levels of speckled activation of caspase-3 in cortical layers 2/3 and 5 compared to those in ethanol-exposed wildtype littermates [22]. However, no significant increase in apoptotic cell death was detected by ethanol exposure in these cilia-deficient mice compared to wild-type littermates. Rather, they exhibited reduced dendritic complexity -fewer dendrites and fewer, shorter branchesassociated with a significant increase in caspase-mediated fragmentation/cleavage of β-actin and α-tubulin cytoskeletal proteins [22]. These findings indicate nonapoptotic effects of increased caspase-3 activation in ethanol-exposed cilia-deficient mice and suggest the novel role of primary cilia in protecting neurons from such adverse effects by ethanol exposure. Activation of the IGF-1 receptor, which is abundantly expressed in the embryonic and postnatal brain and located in primary cilia [62,63], leads to AKT-mediated indirect suppression of caspase activation [64,65]. When cytosolic AKT activation was enhanced by administration of SC79, a small molecule AKT activator [66], 24 h after exposure to ethanol at P7, caspase-3 activation in cortical ciliadeficient mice was significantly reduced in layers 2/3 and 5 [22]. Daily SC79 administration further prevented dendritic atrophy in these mice [22].

Conclusion
Prenatal and early postnatal exposure to ES increases the risk of various neuropsychiatric disorders. For instance, repeated exposure of young animals and children to general anesthesia during the neural vulnerable period results in long-term behavioral and cognitive deficits later in life [16,[67][68][69]. Despite our awareness of this issue, there is still no prescribed treatment for use in combination with anesthesia for infants and children undergoing life-threatening pediatric surgery such as those for congenital heart disease. Exposure to industrial chemicals similarly can have adverse impacts on brain development [70]. Children with fetal alcohol spectrum disorders, caused by prenatal exposure to ethanol due to mother's alcohol drinking during pregnancy, also display a distinct behavioral and cognitive profile that is significantly more severe than children with attention-deficit/hyperactivity disorder [71,72]. However, there remains no effective treatment for the effects caused by such prenatal ES exposure.
The therapeutic compounds shown in Table 1 directly or indirectly inhibit caspase activation and exhibit preventive and ameliorative effects on long-term behavioral deficits when administered before, during, or/and after exposure to ES in animal models. Of these compounds, indirect caspase inhibitors not only inhibit caspase activation but also exhibit a broad spectrum of action. For example, lithium also modulates neurotransmitter signaling [73,74]. Thus, while these indirect caspase inhibitors are effective in modulating multiple pathways involved in the target disease, direct caspase inhibitors are expected to more safely and effectively pinpoint active caspase-mediated events without undesirable effects on other pathways. Such high specificity may be particularly important for the treatment during neural vulnerable period to prevent later disease development.
Nevertheless, it should be noted that, as mentioned earlier, inhibition of normal caspase function, which plays an important role in proper morphogenesis, can have detrimental effects. In the developing brain, it may not only have the direct effect to reduce neuronal apoptosis [23][24][25] but also have indirect effects, such as abnormal vascular density and branching that require proper neurovascular interaction [75]. Further research is needed on the potential problems of inhibiting the caspase pathway during development and how to precisely control this pathway.
Some of the compounds listed in Table 1 have already been used clinically for several distinct diseases; for example, lithium is used to treat symptoms associated with bipolar disorder [44], while melatonin and nicotinamide are used for circadian rhythm sleep disorders [76] and various dermatological indications [77], respectively. Several clinical trials are also ongoing for some of these compounds, including R (+) PPX for obsessive-compulsive disorders [NCT05401019], melatonin for acute ischemic stroke [NCT03843008], and nicotinamide for glioblastoma [NCT04677049].
Therefore, repurposing of these compounds for the treatment of neuropsychiatric disorders caused by perinatal exposure to ES might be worth attempting. Including them, the compounds discussed in this minireview provide hope for potential future development of therapies targeting key molecules and signaling pathways, including the caspase signaling pathways in apoptotic and nonapoptotic processes.