NAD+-Consuming Enzymes in Stem Cell Homeostasis.

Nicotinamide adenine dinucleotide (NAD+) is a coenzyme used in redox reactions, energy metabolism, and mitochondrial biogenesis. NAD+ is also required as a cofactor by nonredox NAD+-dependent enzymes. Hundreds of enzymes that consume NAD+ have been identified. The NAD+-consuming enzymes are involved in a variety of cellular processes such as signal transduction, DNA repair, cellular senescence, and stem cell (SC) homeostasis. In this review, we discussed how different types of NAD+-consuming enzymes regulate SC functions and summarized current research on the roles of the NAD+ consumers in SC homeostasis. We hope to provide a more global and integrative insight to the mechanism and intervention of SC homeostasis via the regulation of the NAD+-consuming enzymes.

Commentary: Novel Cell Culture Paradigm Prolongs Mouse Corneal Epithelial Cell Proliferative Activity In Vitro and In Vivo.

[This corrects the article DOI: 10.3389/fcell.2021.675998.].

Protective Effect of Chlorogenic Acid and Its Analogues on Lead-Induced Developmental Neurotoxicity Through Modulating Oxidative Stress and Autophagy.

Lead (Pb) is among the deleterious heavy metal and has caused global health concerns due to its tendency to cause a detrimental effect on the development of the central nervous system (CNS). Despite being a serious health concern, treatment of Pb poisoning is not yet available, reflecting the pressing need for compounds that can relieve Pb-induced toxicity, especially neurotoxicity. In the quest of exploring protective strategies against Pb-induced developmental neurotoxicity, compounds from natural resources have gained increased attention. Chlorogenic acid (CGA) and its analogues neochlorogenic acid (NCGA) and cryptochlorogenic acid (CCGA) are the important phenolic compounds widely distributed in plants. Herein, utilizing zebrafish as a model organism, we modeled Pb-induced developmental neurotoxicity and investigated the protective effect of CGA, NCGA, and CCGA co-treatment. In zebrafish, Pb exposure (1,000 µg/L) for 5 days causes developmental malformation, loss of dopaminergic (DA) neurons, and brain vasculature, as well as disrupted neuron differentiation in the CNS. Additionally, Pb-treated zebrafish exhibited abnormal locomotion. Notably, co-treatment with CGA (100 µM), NCGA (100 µM), and CCGA (50 µM) alleviated these developmental malformation and neurotoxicity induced by Pb. Further underlying mechanism investigation revealed that these dietary phenolic acid compounds may ameliorate Pb-induced oxidative stress and autophagy in zebrafish, therefore protecting against Pb-induced developmental neurotoxicity. In general, our study indicates that CGA, NCGA, and CCGA could be promising agents for treating neurotoxicity induced by Pb, and CCGA shows the strongest detoxifying activity.

Schaftoside Suppresses Pentylenetetrazol-Induced Seizures in Zebrafish via Suppressing Apoptosis, Modulating Inflammation, and Oxidative Stress.

The lack of disease-modifying therapeutic strategies against epileptic seizures has caused a surge in preclinical research focused on exploring and developing novel therapeutic candidates for epilepsy. Compounds from traditional Chinese medicines (TCMs) have gained much attention for a plethora of neurological diseases, including epilepsy. Herein, for the first time, we evaluated the anticonvulsive effects of schaftoside (SS), a TCM, on pentylenetetrazol (PTZ)-induced epileptic seizures in zebrafish and examined the underlying mechanisms. We observed that SS pretreatments significantly suppressed seizure-like behavior and prolonged the onset of seizures. Zebrafish larvae pretreated with SS demonstrated downregulation of c-fos expression during seizures. PTZ-induced upregulation of apoptotic cells was decreased upon pretreatment with SS. Inflammatory phenomena during seizure progression including the upregulation of interleukin 6 (IL-6), interleukin 1 beta (IL-1ß), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were downregulated upon pretreatment with SS. The PTZ-induced recruitment of immunocytes was in turn reduced upon SS pretreatment. Moreover, SS pretreatment modulated oxidative stress, as demonstrated by decreased levels of catalase (CAT) and increased levels of glutathione peroxidase-1a (GPx1a) and manganese superoxide dismutase (Mn-SOD). However, pretreatment with SS modulated the PTZ-induced downregulation of the relative enzyme activity of CAT, GPx, and SOD. Hence, our findings suggest that SS pretreatment ameliorates PTZ-induced seizures, suppresses apoptosis, and downregulates the inflammatory response and oxidative stress, which potentially protect against further seizures in zebrafish.

Toxicity of different zinc oxide nanomaterials and dose-dependent onset and development of Parkinson's disease-like symptoms induced by zinc oxide nanorods.

With the increasing applications in various fields, the release and accumulation of zinc oxide (ZnO) nanomaterials ultimately lead to unexpected consequences to environment and human health. Therefore, toxicity comparison among ZnO nanomaterials with different shape/size and their adverse effects need better characterization. Here, we utilized zebrafish larvae and human neuroblastoma cells SH-SY5Y to compare the toxic effects of ZnO nanoparticles (ZnO NPs), short ZnO nanorods (s-ZnO NRs), and long ZnO NRs (l-ZnO NRs). We found their developmental- and neuro-toxicity levels were similar, where the smaller sizes showed slightly higher toxicity than the larger sizes. The developmental neurotoxicity of l-ZnO NRs (0.1, 1, 10, 50, and 100 µg/mL) was further investigated since they had the lowest toxicity. Our results indicated that l-ZnO NRs induced developmental neurotoxicity with hallmarks linked to Parkinson's disease (PD)-like symptoms at relatively high doses, including the disruption of locomotor activity as well as neurodevelopmental and PD responsive genes expression, and the induction of dopaminergic neuronal loss and apoptosis in zebrafish brain. l-ZnO NRs activated reactive oxygen species production, whose excessive accumulation triggered mitochondrial damage and mitochondrial apoptosis, eventually leading to PD-like symptoms. Collectively, the developmental- and neuro-toxicity of ZnO nanomaterials was identified, in which l-ZnO NRs harbors a remarkably potential risk for the onset and development of PD at relatively high doses, stressing the discretion of safe range in view of nano-ZnO exposure to ecosystem and human beings.

Synthesis of a novel fluorescent berberine derivative convenient for its subcellular localization study.

Berberine is a naturally occurred isoquinoline alkaloid that shows great potential for developing anticancer drugs. However, the problem stays of poor understanding of the mechanisms of anticancer action of berberine. It depends on evaluation of berberine's pharmacokinetics, namely monitoring of its uptake and distribution in cells, tissues and organs. In order to address these problems, we have designed and synthesized a novel berberine derivative BBR-BODIPY bearing a fluorescent tag that allows screening its interaction with the targeted cells. It was shown that the synthesized fluorescent derivative could penetrate into human breast carcinoma MCF7 cells, and then induced apoptosis detected by the Western Blot analysis as changed expression of apoptosis-related proteins, including Bax, Bcl2, and Cyto C released from mitochondria, Cleaved Caspase 9, Cleaved PARP, Pro-Caspase 3, and Cleaved Caspase 3. The results of MitoTracker analysis followed by the confocal microscopy of sub-cellular localization of BBR-BODIPY in the MCF7 cells demonstrated excellent cell-penetrating ability of this compound even at low concentrations, and mitochondria was the main site of its accumulation. Together with the results of Western Blot analysis, these data indicated that the mitochondria pathway might be involved in berberine-induced apoptosis.

α-asarone induces cardiac defects and QT prolongation through mitochondrial apoptosis pathway in zebrafish.

α-asarone is a natural phenylpropene found in several plants, which are widely used for flavoring foods and treating diseases. Previous studies have demonstrated that α-asarone has many pharmacological functions, while some reports indicated its toxicity. However, little is known about its cardiovascular effects. This study investigated developmental toxicity of α-asarone in zebrafish, especially the cardiotoxicity. Zebrafish embryos were exposed to different concentrations of α-asarone (1, 3, 5, 10, and 30 µM). Developmental toxicity assessments revealed that α-asarone did not markedly affect mortality and hatching rate. In contrast, there was a concentration-dependent increase in malformation rate of zebrafish treated with α-asarone. The most representative cardiac defects were increased heart malformation rate, pericardial edema areas, sinus venosus-bulbus arteriosus distance, and decreased heart rate. Notably, we found that α-asarone impaired the cardiac function of zebrafish by prolonging the mean QTc duration and causing T-wave abnormalities. The expressions of cardiac development-related key transcriptional regulators tbx5, nkx2.5, hand2, and gata5 were all changed under α-asarone exposure. Further investigation addressing the mechanism indicated that α-asarone triggered apoptosis mainly in the heart region of zebrafish. Moreover, the elevated expression of puma, cyto C, afap1, caspase 3, and caspase 9 in treated zebrafish suggested that mitochondrial apoptosis is likely to be the main reason for α-asarone induced cardiotoxicity. These findings revealed the cardiac developmental toxicity of α-asarone, expanding our knowledge about the toxic effect of α-asarone on living organisms.

Anti-Inflammation Associated Protective Mechanism of Berberine and its Derivatives on Attenuating Pentylenetetrazole-Induced Seizures in Zebrafish.

Epileptic seizures are characterized by synchronized discharges of neurons, leading to the activation of inflammatory responses that in turn contributes to seizure progression. Berberine (BBR), a bioactive constituent extracted from berberis, has been known to relieve seizures in rodent models. In this study, we synthesized two derivatives of berberine (BBR-D1 and BBR-D2) to compare their seizure reducing effect with BBR in pentylenetetrazole (PTZ)-induced seizures in zebrafish. We found a structure-activity relationship between hydrophilic/hydrophobic composition of the derivatives and their anticonvulsant activity. We also investigated the underlying mechanism related to their anti-inflammatory effect during seizures. BBR and its derivatives increased the seizure onset latency and suppressed the seizure-like behavior after PTZ treatment. Zebrafish larvae pretreated with BBR and its derivatives showed recovery on c-fos expression and neuronal discharges during seizures. The inflammatory responses occurred during the progression of seizures, including the recruitment of macrophages and neutrophils as well as an up-regulation of tumor necrosis factor alpha (TNFα), interleukin 1 beta (il1ß), and interleukin 6 (il6). This effect was significantly suppressed by the pretreatment of BBR and its derivatives. Our results suggest that BBR and its derivatives attenuate PTZ-induced seizures and modulate anti-inflammatory effect to potentially protect zebrafish from the occurrence of further seizures. From the tested compounds, BBR-D1 (the hydrophilic berberrubine) showed the strongest seizure reducing effect. Graphical Abstract Two derivatives of berberine (BBR-D1 and BBR-D2) were synthesized to compare their seizure reducing effect with BBR in pentylenetetrazole (PTZ)-induced seizures in zebrafish. BBR and its derivatives increased the seizure onset latency and suppressed the seizure-like behavior after PTZ treatment. Zebrafish larvae pretreated with BBR and its derivatives showed recovery on c-fos expression and neuronal discharges during seizures. The inflammatory responses occurred during the progression of seizures, including the recruitment of macrophages and neutrophils as well as an up-regulation of tumor necrosis factor alpha (TNFα), interleukin 1 beta (il1ß), and interleukin 6 (il6). This effect was significantly suppressed by the pretreatment of BBR and its derivatives.

Zebrafish behavioral phenomics employed for characterizing behavioral neurotoxicity caused by silica nanoparticles.

Nowadays, silica nanoparticles (SiNPs) as one of the most productive nano-powder, has been extensively applied in various filed. The potential harm of SiNPs has previously received severe attention. A bulk of researches have proven the adverse effect of SiNPs on the health of ecological organisms and human. However, neurotoxic impacts of SiNPs, still remain in the stage of exploration. The potential neurotoxic effects of SiNPs need to be further explored. And the toxic mechanism needs comprehensive clarification. Herein, the neurotoxicity of SiNPs of various concentrations (100, 300, 1000 µg/mL) on adult zebrafish was determined by behavioral phenotyping and confirmed by molecular biology techniques such as qPCR. Behavioral phenotype revealed observable effects of SiNPs on disturbing light/dark preference, dampening exploratory behavior, inhibiting memory capability. Furthermore, the relationship between neurotoxic symptom and the transcriptional alteration of autophagy- and parkinsonism-related genes was preliminarily assessed. Importantly, further investigations should be carried out to determine the effects of SiNPs to cause neurodegeneration in the brain as well as to decipher the specific neurotoxic mechanisms. In sum, this work comprehensively evaluated the neurotoxic effect of small-sized SiNPs on overall neurobehavioral profiles and indicated the potential for SiNPs to cause Parkinson's disease, which will provide a solid reference for the research on the neurotoxicity of SiNPs.

Zebrafish neurobehavioral phenomics applied as the behavioral warning methods for fingerprinting endocrine disrupting effect by lead exposure at environmentally relevant level.

Environmental lead (Pb) exposure is a great hazard to the public health. Although environmentally relevant Pb poisoning is preventable, insidious Pb contaminants are still a major threat to human health. Herein, we reported that exposure to Pb at environmentally relevant concentration level (1 µg/L, 10 µg/L and 100 µg/L), disturbed the courtship behavior of adult male zebrafish and further altered the transcriptional patterns of key genes involved in testicular steroidogenesis (igf3, amh, piwil1, lhcgr, fshr, cyp11c1, star, cyp19a1a, cyp19a1b) and apoptosis (bax, cytoC, caspase 9, caspase 3, puma). Both the behavioral and the transcriptional profiles share a similar biphasic dose response, with stimulatory effects after low-level exposure and inhibitory effects after high-level exposure. This results revealed the endocrine disrupting effects of Pb even at an environmentally relevant level within the concentration range of ambient water quality criteria (AWQC) and the reliability of locomotion fingerprint as the indicator for detecting the risk induced by Pb pollution. Current research, for the first time, employed the ZebraTower system as the biological early warning system (BEWS) to find that Pb exerted biphasic effects on the courtship behavior and endocrine regulation of male adult zebrafish. Methodologically, we firstly propose an efficient solution to monitor and assess the risk of Pb exposure by combining the (BEWS) and data analyzing methods such as zebrafish phenomics, which would make a contribution to the detection and prevention of environmentally relevant Pb poisoning.

Zebrafish behavioral phenomics applied for phenotyping aquatic neurotoxicity induced by lead contaminants of environmentally relevant level.

Environmental lead (Pb) exposure is a worldwide threat due to the ubiquitous contamination. Although the adverse effects of Pb on human health have previously been extensively explored, the eco-toxicological effects on aquatic vertebrates still need further investigation. In addition, there is a paucity in the knowledge of behavioral and physiological effects of Pb within the range of environmental relevant concentration (under 100 µg/L) on aquatic organisms such as zebrafish. Herein, we demonstrated that adult male zebrafish (Danio rerio) exposed to Pb at environmental concentration level (1 µg/L, 10 µg/L and 100 µg/L) for 14 days, exhibited obvious neuro-behavioral alteration including disturbed light dark preference, impaired exploratory behaviors and inhibited spatial working memory. The alteration of entire behavioral profiles was further associated with the disturbed expression patterns of mRNA level of key genes involved in neurodevelopment (gap43, syn2a, th, dat, and drd1b), neurotoxic effects (c-fos and gfap), and stress responses (tap, mt1, hsp70, and hsp90). To determine the comprehensively effect of aquatic contaminants on the entire behavioral profiles, behavioral phenomic data were obtained by hierarchical clustering analysis. Overall, we employed behavioral phenomics methods to find that Pb within standard chronic Pb toxic criteria, significantly altered behavioral phenotype and brain physiology, which would exert profound ecological consequences and offer the reference for adjustment of aquatic toxic criteria.

Synergistic effects of Pb and repeated heat pulse on developmental neurotoxicity in zebrafish.

Pollutant discharges to the aquatic environment often contain multiple environmental stressors, affecting aquatic organisms. To mimic the discharges from nuclear and industry facilities, the combined effects of two independent types of stressors, heavy metal Pb and repeated heat pulse were addressed in this study. We investigated the developmental toxicity of combined treatment, especially its toxic effects on zebrafish neurodevelopment. The normal embryos at 4 hpf were exposed to 0.2 mM of Pb dissolved in the bathing medium with different temperatures (30, 32, and 34 °C) and then maintained in an incubator at 28 °C. After performing above treatment once every 24 h for 6 days, we found that combined treatment significantly affected neural development, including loss of dopaminergic (DA) neurons and brain vasculature, disruption of locomotor activity and neurodevelopmental genes expression in a temperature-dependent manner as compared to the Pb alone exposure group, indicating that repeated heat pulse enhances these negative impacts induced by Pb. In contrast, no apparent toxicity was observed in repeated heat pulse alone groups, suggesting that Pb treatment reduces thermal tolerance in zebrafish, which emphasized the importance to evaluate synergistic effects of Pb and repeated heat pulse. Moreover, repeated heat pulse aggravated Pb-induced apoptosis in the zebrafish brain. Further study of the underlying mechanism suggested that Caspase 3 regulated apoptosis was involved in this process. Taken together, our findings shed light on the full understanding of toxic effects of discharges from industrial applications on living organisms and its environmental impact.

Activation of BDNF-TrkB signaling pathway-regulated brain inflammation in pentylenetetrazole-induced seizures in zebrafish.

Seizures are sustained neuronal hyperexcitability in brain that result in loss of consciousness and injury. Understanding how the brain responds to seizures is critical to help developing new therapeutic strategies for epilepsy, a neurological disorder characterized by recurrent and unprovoked seizures. However, the mechanisms underlying seizure-dependent alterations of biological properties are poorly understood. In this study, we analyzed gene expression profiles of the zebrafish heads that were undergoing seizures and identified 1776 differentially expressed genes. Gene-regulatory network analysis revealed that BDNF-TrkB signaling pathway positively regulated brain inflammation in zebrafish during seizures. Using K252a, a TrkB inhibitor to block BDNF-TrkB signaling pathway, attenuated pentylenetetrazole (PTZ)-induced seizures, which also confirmed BDNF-TrkB mediated inflammatory responses including regulation of il1ß and nfκb, and neutrophil and macrophage infiltration of brain. Our results have provided novel insights into seizure-induced brain inflammation in zebrafish and anti-inflammatory related therapy for epilepsy.