Human Oligodendrocytes and Myelin In Vitro to Evaluate Developmental Neurotoxicity

Neurodevelopment is uniquely sensitive to toxic insults and there are concerns that environmental chemicals are contributing to widespread subclinical developmental neurotoxicity (DNT). Increased DNT evaluation is needed due to the lack of such information for most chemicals in common use, but in vivo studies recommended in regulatory guidelines are not practical for the large-scale screening of potential DNT chemicals. It is widely acknowledged that developmental neurotoxicity is a consequence of disruptions to basic processes in neurodevelopment and that testing strategies using human cell-based in vitro systems that mimic these processes could aid in prioritizing chemicals with DNT potential. Myelination is a fundamental process in neurodevelopment that should be included in a DNT testing strategy, but there are very few in vitro models of myelination. Thus, there is a need to establish an in vitro myelination assay for DNT. Here, we summarize the routes of myelin toxicity and the known models to study this particular endpoint.     

Developmental Exposure to Low Concentrations of Methylmercury Causes Increase in Anxiety-Related Behaviour and Locomotor Impairments in Zebrafish

Methylmercury (MeHg) is a ubiquitous pollutant shown to cause developmental neurotoxicity, even at low levels. However, there is still a large gap in our understanding of the mechanisms linking early-life exposure to life-long behavioural impairments. Our aim was to characterise the short- and long-term effects of developmental exposure to low doses of MeHg on anxiety-related behaviours in zebrafish, and to test the involvement of neurological pathways related to stress-response. Zebrafish embryos were exposed to sub-acute doses of MeHg (0, 5, 10, 15, 30 nM) throughout embryo-development, and tested for anxiety-related behaviours and locomotor activity at larval (light/dark locomotor activity) and adult (novel tank and tap assays) life-stages. Exposure to all doses of MeHg caused increased anxiety-related responses; heightened response to the transition from light to dark in larvae, and a stronger dive response in adults. In addition, impairment in locomotor activity was observed in the higher doses in both larvae and adults. Finally, the expressions of several neural stress-response genes from the HPI-axis and dopaminergic system were found to be disrupted in both life-stages. Our results provide important insights into dose-dependent differences in exposure outcomes, the development of delayed effects over the life-time of exposed individuals and the potential mechanisms underlying these effects.     

Knockout of Katnal2 Leads to Autism-like Behaviors and Developmental Delay in Zebrafish

KATNAL2 mutations have been associated with autism spectrum disorder (ASD) and other related neurodevelopmental disorders (NDDs) such as intellectual disability (ID) in several cohorts. KATNAL2 has been implicated in brain development, as it is required for ciliogenesis in Xenopus and is required for dendritic arborization in mice. However, a causative relationship between the disruption of Katnal2 function and behavioral defects has not been established. Here, we generated a katnal2 null allele in zebrafish using CRISPR/Cas9-mediated genome editing and carried out morphological and behavioral characterizations. We observed that katnal2^-/- embryos displayed delayed embryonic development especially during the convergence and extension (CE) movement. The hatched larvae showed reduced brain size and body length. In the behavioral tests, the katnal2^-/- zebrafish exhibited reduced locomotor activity both in larvae and adults; increased nocturnal waking activity in larvae; and enhanced anxiety-like behavior, impaired social interaction, and reduced social cohesion in adults. These findings indicate an important role for katnal2 in development and behavior, providing an in vivo model to study the mechanisms underlying the ASD related to KATNAL2 mutations.     

Ipconazole Disrupts Mitochondrial Homeostasis and Alters GABAergic Neuronal Development in Zebrafish

Ipconazole, a demethylation inhibitor of fungal ergosterol biosynthesis, is widely used in modern agriculture for foliar and seed treatment, and is authorized for use in livestock feed. Waste from ipconazole treatment enters rivers and groundwater through disposal and rain, posing potential toxicity to humans and other organisms. Its metabolites remain stable under standard hydrolysis conditions; however, their neurodevelopmental toxicity is unknown. We investigated the potential neurodevelopmental toxicity of ipconazole pesticides in zebrafish (Danio rerio). Our behavioral monitoring demonstrated that the locomotive activity of ipconazole-exposed zebrafish larvae was reduced during early development, even when morphological abnormalities were undetected. Molecular profiling demonstrated that the mitochondrial-specific antioxidants, superoxide dismutases 1 and 2, and the genes essential for mitochondrial genome maintenance and functions were specifically reduced in ipconazole-treated (0.02 μg/mL) embryos, suggesting underlying ipconazole-driven oxidative stress. Consistently, ipconazole treatment substantially reduced hsp70 expression and increased ERK1/2 phosphorylation in a dose-dependent manner. Interrupted gad1b expression confirmed that GABAergic inhibitory neurons were dysregulated at 0.02 μg/mL ipconazole, whereas glutamatergic excitatory and dopaminergic neurons remained unaffected, resulting in an uncoordinated neural network. Additionally, ipconazole-treated (2 μg/mL) embryos exhibited caspase-independent cell death. This suggests that ipconazole has the potential to alter neurodevelopment by dysregulating mitochondrial homeostasis.     

Modelling Autism Spectrum Disorder (ASD) and Attention-Deficit/Hyperactivity Disorder (ADHD) Using Mice and Zebrafish

Autism spectrum disorders (ASD) and attention-deficit/hyperactivity disorder (ADHD) are two debilitating neurodevelopmental disorders. The former is associated with social impairments whereas the latter is associated with inattentiveness, hyperactivity, and impulsivity. There is recent evidence that both disorders are somehow related and that genes may play a large role in these disorders. Despite mounting human and animal research, the neurological pathways underlying ASD and ADHD are still not well understood. Scientists investigate neurodevelopmental disorders by using animal models that have high similarities in genetics and behaviours with humans. Mice have been utilized in neuroscience research as an excellent animal model for a long time; however, the zebrafish has attracted much attention recently, with an increasingly large number of studies using this model. In this review, we first discuss ASD and ADHD aetiology from a general point of view to their characteristics and treatments. We also compare mice and zebrafish for their similarities and discuss their advantages and limitations in neuroscience. Finally, we summarize the most recent and existing research on zebrafish and mouse models of ASD and ADHD. We believe that this review will serve as a unique document providing interesting information to date about these models, thus facilitating research on ASD and ADHD.     

除草剂对斑马鱼毒性效应研究概述

除草剂的大量、不合理使用对水体造成了严重污染,威胁水生生物的生存及人类健康。不少研究者开始对除草剂生态环境风险评估进行研究。斑马鱼因其自身独特的优势而已成为国际公认的模式脊椎动物之一,是污染物风险评估选用的绝佳材料之一。对斑马鱼在风险评估中的地位、除草剂对斑马鱼及其胚胎的毒性与富集进行了综述。     

Developmental Toxicity and Biotransformation of Two Anti-Epileptics in Zebrafish Embryos and Early Larvae

The zebrafish (Danio rerio) embryo is gaining interest as a bridging tool between in-vitro and in-vivo developmental toxicity studies. However, cytochrome P450 (CYP)-mediated drug metabolism in this model is still under debate. Therefore, we investigated the potential of zebrafish embryos and larvae to bioactivate two known anti-epileptics, carbamazepine (CBZ) and phenytoin (PHE), to carbamazepine-10,11-epoxide (E-CBZ) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), respectively. First, zebrafish were exposed to CBZ, PHE, E-CBZ and HPPH from 5¼- to 120-h post fertilization (hpf) and morphologically evaluated. Second, the formations of E-CBZ and HPPH were assessed in culture medium and in whole-embryo extracts at different time points by targeted LC-MS. Finally, E-CBZ and HPPH formation was also assessed in adult zebrafish liver microsomes and compared with those of human, rat, and rabbit. The present study showed teratogenic effects for CBZ and PHE, but not for E-CBZ and HPPH. No HPPH was detected during organogenesis and E-CBZ was only formed at the end of organogenesis. E-CBZ and HPPH formation was also very low-to-negligible in adult zebrafish compared with the mammalian species. As such, other metabolic pathways than those of mammals are involved in the bioactivation of CBZ and PHE, or, these anti-epileptics are teratogens and do not require bioactivation in the zebrafish.     

Zebrafish and Medaka: Important Animal Models for Human Neurodegenerative Diseases

Animal models of human neurodegenerative disease have been investigated for several decades. In recent years, zebrafish (Danio rerio) and medaka (Oryzias latipes) have become popular in pathogenic and therapeutic studies about human neurodegenerative diseases due to their small size, the optical clarity of embryos, their fast development, and their suitability to large-scale therapeutic screening. Following the emergence of a new generation of molecular biological technologies such as reverse and forward genetics, morpholino, transgenesis, and gene knockout, many human neurodegenerative disease models, such as Parkinson’s, Huntington’s, and Alzheimer’s, were constructed in zebrafish and medaka. These studies proved that zebrafish and medaka genes are functionally conserved in relation to their human homologues, so they exhibit similar neurodegenerative phenotypes to human beings. Therefore, fish are a suitable model for the investigation of pathologic mechanisms of neurodegenerative diseases and for the large-scale screening of drugs for potential therapy. In this review, we summarize the studies in modelling human neurodegenerative diseases in zebrafish and medaka in recent years.     

Molecular Mechanism of Arsenic-Induced Neurotoxicity including Neuronal Dysfunctions

Arsenic is a key environmental toxicant having significant impacts on human health. Millions of people in developing countries such as Bangladesh, Mexico, Taiwan, and India are affected by arsenic contamination through groundwater. Environmental contamination of arsenic leads to leads to various types of cancers, coronary and neurological ailments in human. There are several sources of arsenic exposure such as drinking water, diet, wood preservatives, smoking, air and cosmetics, while, drinking water is the most explored route. Inorganic arsenic exhibits higher levels of toxicity compared its organic forms. Exposure to inorganic arsenic is known to cause major neurological effects such as cytotoxicity, chromosomal aberration, damage to cellular DNA and genotoxicity. On the other hand, long-term exposure to arsenic may cause neurobehavioral effects in the juvenile stage, which may have detrimental effects in the later stages of life. Thus, it is important to understand the toxicology and underlying molecular mechanism of arsenic which will help to mitigate its detrimental effects. The present review focuses on the epidemiology, and the toxic mechanisms responsible for arsenic induced neurobehavioral diseases, including strategies for its management from water, community and household premises. The review also provides a critical analysis of epigenetic and transgenerational modifications, mitochondrial oxidative stress, molecular mechanisms of arsenic-induced oxidative stress, and neuronal dysfunction.     

Inner Ear and Muscle Developmental Defects in Smpx-Deficient Zebrafish Embryos

The last decade has witnessed the identification of several families affected by hereditary non-syndromic hearing loss (NSHL) caused by mutations in the SMPX gene and the loss of function has been suggested as the underlying mechanism. In the attempt to confirm this hypothesis we generated an Smpx-deficient zebrafish model, pointing out its crucial role in proper inner ear development. Indeed, a marked decrease in the number of kinocilia together with structural alterations of the stereocilia and the kinocilium itself in the hair cells of the inner ear were observed. We also report the impairment of the mechanotransduction by the hair cells, making SMPX a potential key player in the construction of the machinery necessary for sound detection. This wealth of evidence provides the first possible explanation for hearing loss in SMPX-mutated patients. Additionally, we observed a clear muscular phenotype consisting of the defective organization and functioning of muscle fibers, strongly suggesting a potential role for the protein in the development of muscle fibers. This piece of evidence highlights the need for more in-depth analyses in search for possible correlations between SMPX mutations and muscular disorders in humans, thus potentially turning this non-syndromic hearing loss-associated gene into the genetic cause of dysfunctions characterized by more than one symptom, making SMPX a novel syndromic gene.     

Zebrafish,Medaka and Turquoise Killifish for Understanding Human Neurodegenerative/Neurodevelopmental Disorders

In recent years, small fishes such as zebrafish and medaka have been widely recognized as model animals. They have high homology in genetics and tissue structure with humans and unique features that mammalian model animals do not have, such as transparency of embryos and larvae, a small body size and ease of experiments, including genetic manipulation. Zebrafish and medaka have been used extensively in the field of neurology, especially to unveil the mechanisms of neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease, and recently, these fishes have also been utilized to understand neurodevelopmental disorders such as autism spectrum disorder. The turquoise killifish has emerged as a new and unique model animal, especially for ageing research due to its unique life cycle, and this fish also seems to be useful for age-related neurological diseases. These small fishes are excellent animal models for the analysis of human neurological disorders and are expected to play increasing roles in this field. Here, we introduce various applications of these model fishes to improve our understanding of human neurological disorders.     

Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes

CRISPR-Cas9-based genome-editing is a highly efficient and cost-effective method to generate zebrafish loss-of-function alleles. However, introducing patient-specific variants into the zebrafish genome with CRISPR-Cas9 remains challenging. Targeting options can be limited by the predetermined genetic context, and the efficiency of the homology-directed DNA repair pathway is relatively low. Here, we illustrate our efficient approach to develop knock-in zebrafish models using two previously variants associated with hereditary sensory deficits. We employ sgRNA-Cas9 ribonucleoprotein (RNP) complexes that are micro-injected into the first cell of fertilized zebrafish eggs together with an asymmetric, single-stranded DNA template containing the variant of interest. The introduction of knock-in events was confirmed by massive parallel sequencing of genomic DNA extracted from a pool of injected embryos. Simultaneous morpholino-induced blocking of a key component of the non-homologous end joining DNA repair pathway, Ku70, improved the knock-in efficiency for one of the targets. Our use of RNP complexes provides an improved knock-in efficiency as compared to previously published studies. Correct knock-in events were identified in 3–8% of alleles, and 30–45% of injected animals had the target variant in their germline. The detailed technical and procedural insights described here provide a valuable framework for the efficient development of knock-in zebrafish models.     

Efficient Neuroprotective Rescue of Sacsin-Related Disease Phenotypes in Zebrafish

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a multisystem hereditary ataxia associated with mutations in SACS, which encodes sacsin, a protein of still only partially understood function. Although mouse models of ARSACS mimic largely the disease progression seen in humans, their use in the validation of effective therapies has not yet been proposed. Recently, the teleost Danio rerio has attracted increasing attention as a vertebrate model that allows rapid and economical screening, of candidate molecules, and thus combines the advantages of whole-organism phenotypic assays and in vitro high-throughput screening assays. Through CRISPR/Cas9-based mutagenesis, we generated and characterized a zebrafish sacs-null mutant line that replicates the main features of ARSACS. The sacs-null fish showed motor impairment, hindbrain atrophy, mitochondrial dysfunction, and reactive oxygen species accumulation. As proof of principle for using these mutant fish in high-throughput screening studies, we showed that both acetyl-DL-leucine and tauroursodeoxycholic acid improved locomotor and biochemical phenotypes in sacs^−/− larvae treated with these neuroprotective agents, by mediating significant rescue of the molecular functions altered by sacsin loss. Taken together, the evidence here reported shows the zebrafish to be a valuable model organism for the identification of novel molecular mechanisms and for efficient and rapid in vivo optimization and screening of potential therapeutic compounds. These findings may pave the way for new interventions targeting the earliest phases of Purkinje cell degeneration in ARSACS.     

Assessing the Morphological and Behavioral Toxicity of Catechol Using Larval Zebrafish

Catechol is a ubiquitous chemical used in the manufacturing of fragrances, pharmaceuticals and flavorants. Environmental exposure occurs in a variety of ways through industrial processes, during pyrolysis and in effluent, yet despite its prevalence, there is limited information regarding its toxicity. While the genotoxicity and gastric carcinogenicity of catechol have been described in depth, toxicological studies have potentially overlooked a number of other effects relevant to humans. Here, we have made use of a general and behavioral larval zebrafish toxicity assay to describe previously unknown catechol-based toxicological phenomena. Behavioral testing revealed catechol-induced hypoactivity at concentrations an order of magnitude lower than observable endpoints. Catechol exposure also resulted in punctate melanocytes with concomitant decreases in the expression of pigment production and regulation markers mitfa, mc1r and tyr. Because catechol is converted into a number of toxic metabolites by tyrosinase, an enzyme found almost exclusively in melanocytes, an evaluation of the effects of catechol on these cells is critical to evaluating the safety of this chemical. This work provides insights into the toxic nature of catechol and highlights the benefits of the zebrafish larval testing platform in being able to dissect multiple aspects of toxicity with one model.     

Damage to Olfactory Organs of Adult Zebrafish Induced by Diesel Particulate Matter

Particulate matter (PM) is an environmental hazard that is associated with various human health risks. The olfactory system is directly exposed to PM; therefore, the influence of PM exposure on olfactory function must be investigated. In this study, we propose a zebrafish olfactory model to evaluate the effects of exposure to diesel particulate matter (DPM), which was labeled Korean diesel particulate matter (KDP20). KDP20 comprises heavy metals and polycyclic aromatic hydrocarbons (PAHs). KDP20 exposed olfactory organs exhibited reduced cilia and damaged epithelium. Olfactory dysfunction was confirmed using an odor-mediated behavior test. Furthermore, the olfactory damage was analyzed using Alcian blue and anti-calretinin staining. KDP20 exposed olfactory organs exhibited histological damages, such as increased goblet cells, decreased cell density, and calretinin level. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed that PAHs exposure related genes (AHR2 and CYP1A) were upregulated. Reactive oxidation stress (ROS) (CAT) and inflammation (IL-1B) related genes were upregulated. Furthermore, olfactory sensory neuron (OSN) related genes (OMP and S100) were downregulated. In conclusion, KDP20 exposure induced dysfunction of the olfactory system. Additionally, the zebrafish olfactory system exhibited a regenerative capacity with recovery conditions. Thus, this model may be used in future investigating PM-related diseases.     

应用胚胎检测技术评价氟吗啉对斑马鱼胚胎-幼体发育的影响

采用斑马鱼胚胎发育技术，通过对斑马鱼急性毒性及胚胎时期的鱼卵进行染毒来观测孵化率、畸形率及死亡率，由此评价氟吗啉对斑马鱼胚胎-幼体发育的影响。试验结果表明，氟吗啉对斑马鱼胚胎及成鱼这一段时期的影响较小，根据国家环保局对水生生物的毒性划分标准，氟吗啉的毒性为“低级毒”。     

Long-Chain Acyl-Carnitines Interfere with Mitochondrial ATP Production Leading to Cardiac Dysfunction in Zebrafish

In the human heart, the energy supplied by the production of ATP is predominately accomplished by ß-oxidation in mitochondria, using fatty acids (FAs) as the primary fuel. Long-chain acylcarnitines (LCACs) are intermediate forms of FA transport that are essential for FA delivery from the cytosol into mitochondria. Here, we analyzed the impact of the LCACs C18 and C18:1 on mitochondrial function and, subsequently, on heart functionality in the in vivo vertebrate model system of zebrafish (Danio rerio). Since LCACs are formed and metabolized in mitochondria, we assessed mitochondrial morphology, structure and density in C18- and C18:1-treated zebrafish and found no mitochondrial alterations compared to control-treated (short-chain acylcarnitine, C3) zebrafish embryos. However, mitochondrial function and subsequently ATP production was severely impaired in C18- and C18:1-treated zebrafish embryos. Furthermore, we found that C18 and C18:1 treatment of zebrafish embryos led to significantly impaired cardiac contractile function, accompanied by reduced heart rate and diminished atrial and ventricular fractional shortening, without interfering with cardiomyocyte differentiation, specification and growth. In summary, our findings provide insights into the direct role of long-chain acylcarnitines on vertebrate heart function by interfering with regular mitochondrial function and thereby energy allocation in cardiomyocytes.     

Toxicological Profile of Plasmonic Nanoparticles in Zebrafish Model

Plasmonic nanoparticles are increasingly employed in several fields, thanks to their unique, promising properties. In particular, these particles exhibit a surface plasmon resonance combined with outstanding absorption and scattering properties. They are also easy to synthesize and functionalize, making them ideal for nanotechnology applications. However, the physicochemical properties of these nanoparticles can make them potentially toxic, even if their bulk metallic forms are almost inert. In this review, we aim to provide a more comprehensive understanding of the potential adverse effects of plasmonic nanoparticles in zebrafish (Danio rerio) during both development and adulthood, focusing our attention on the most common materials used, i.e., gold and silver.     

Zebrafish: A New Promise to Study the Impact of Metabolic Disorders on the Brain

Zebrafish has become a popular model to study many physiological and pathophysiological processes in humans. In recent years, it has rapidly emerged in the study of metabolic disorders, namely, obesity and diabetes, as the regulatory mechanisms and metabolic pathways of glucose and lipid homeostasis are highly conserved between fish and mammals. Zebrafish is also widely used in the field of neurosciences to study brain plasticity and regenerative mechanisms due to the high maintenance and activity of neural stem cells during adulthood. Recently, a large body of evidence has established that metabolic disorders can alter brain homeostasis, leading to neuro-inflammation and oxidative stress and causing decreased neurogenesis. To date, these pathological metabolic conditions are also risk factors for the development of cognitive dysfunctions and neurodegenerative diseases. In this review, we first aim to describe the main metabolic models established in zebrafish to demonstrate their similarities with their respective mammalian/human counterparts. Then, in the second part, we report the impact of metabolic disorders (obesity and diabetes) on brain homeostasis with a particular focus on the blood–brain barrier, neuro-inflammation, oxidative stress, cognitive functions and brain plasticity. Finally, we propose interesting signaling pathways and regulatory mechanisms to be explored in order to better understand how metabolic disorders can negatively impact neural stem cell activity.