Genetic Modeling of the Neurodegenerative Disease Spinocerebellar Ataxia Type 1 in Zebrafish

Dominant spinocerebellar ataxias (SCAs) are progredient neurodegenerative diseases commonly affecting the survival of Purkinje cells (PCs) in the human cerebellum. Spinocerebellar ataxia type 1 (SCA1) is caused by the mutated ataxin1 (Atx1) gene product, in which a polyglutamine stretch encoded by CAG repeats is extended in affected SCA1 patients. As a monogenetic disease with the Atx1-polyQ protein exerting a gain of function, SCA1 can be genetically modelled in animals by cell type-specific overexpression. We have established a transgenic PC-specific SCA1 model in zebrafish coexpressing the fluorescent reporter protein mScarlet together with either human wild type Atx1[30Q] as control or SCA1 patient-derived Atx1[82Q]. SCA1 zebrafish display an age-dependent PC degeneration starting at larval stages around six weeks postfertilization, which continuously progresses during further juvenile and young adult stages. Interestingly, PC degeneration is observed more severely in rostral than in caudal regions of the PC population. Although such a neuropathology resulted in no gross locomotor control deficits, SCA1-fish with advanced PC loss display a reduced exploratory behaviour. In vivo imaging in this SCA1 model may help to better understand such patterned PC death known from PC neurodegeneration diseases, to elucidate disease mechanisms and to provide access to neuroprotective compound characterization in vivo.     

Disease Modeling of Rare Neurological Disorders in Zebrafish

Rare diseases are those which affect a small number of people compared to the general population. However, many patients with a rare disease remain undiagnosed, and a large majority of rare diseases still have no form of viable treatment. Approximately 40% of rare diseases include neurologic and neurodevelopmental disorders. In order to understand the characteristics of rare neurological disorders and identify causative genes, various model organisms have been utilized extensively. In this review, the characteristics of model organisms, such as roundworms, fruit flies, and zebrafish, are examined, with an emphasis on zebrafish disease modeling in rare neurological disorders.     

Developmental and Neurotoxicity of Acrylamide to Zebrafish

Acrylamide is a commonly used industrial chemical that is known to be neurotoxic to mammals. However, its developmental toxicity is rarely assessed in mammalian models because of the cost and complexity involved. We used zebrafish to assess the neurotoxicity, developmental and behavioral toxicity of acrylamide. At 6 h post fertilization, zebrafish embryos were exposed to four concentrations of acrylamide (10, 30, 100, or 300 mg/L) in a medium for 114 h. Acrylamide caused developmental toxicity characterized by yolk retention, scoliosis, swim bladder deficiency, and curvature of the body. Acrylamide also impaired locomotor activity, which was measured as swimming speed and distance traveled. In addition, treatment with 100 mg/L acrylamide shortened the width of the brain and spinal cord, indicating neuronal toxicity. In summary, acrylamide induces developmental toxicity and neurotoxicity in zebrafish. This can be used to study acrylamide neurotoxicity in a rapid and cost-efficient manner.     

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.     

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.     

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.     

Probiotics Enhance Bone Growth and Rescue BMP Inhibition: New Transgenic Zebrafish Lines to Study Bone Health

Zebrafish larvae, especially gene-specific mutants and transgenic lines, are increasingly used to study vertebrate skeletal development and human pathologies such as osteoporosis, osteopetrosis and osteoarthritis. Probiotics have been recognized in recent years as a prophylactic treatment for various bone health issues in humans. Here, we present two new zebrafish transgenic lines containing the coding sequences for fluorescent proteins inserted into the endogenous genes for sp7 and col10a1a with larvae displaying fluorescence in developing osteoblasts and the bone extracellular matrix (mineralized or non-mineralized), respectively. Furthermore, we use these transgenic lines to show that exposure to two different probiotics, Bacillus subtilis and Lactococcus lactis, leads to an increase in osteoblast formation and bone matrix growth and mineralization. Gene expression analysis revealed the effect of the probiotics, particularly Bacillus subtilis, in modulating several skeletal development genes, such as runx2, sp7, spp1 and col10a1a, further supporting their ability to improve bone health. Bacillus subtilis was the more potent probiotic able to significantly reverse the inhibition of bone matrix formation when larvae were exposed to a BMP inhibitor (LDN212854).     

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。     

A Zebrafish Model for a Rare Genetic Disease Reveals a Conserved Role for FBXL3 in the Circadian Clock System

The circadian clock, which drives a wide range of bodily rhythms in synchrony with the day–night cycle, is based on a molecular oscillator that ticks with a period of approximately 24 h. Timed proteasomal degradation of clock components is central to the fine-tuning of the oscillator’s period. FBXL3 is a protein that functions as a substrate-recognition factor in the E3 ubiquitin ligase complex, and was originally shown in mice to mediate degradation of CRY proteins and thus contribute to the mammalian circadian clock mechanism. By exome sequencing, we have identified a FBXL3 mutation in patients with syndromic developmental delay accompanied by morphological abnormalities and intellectual disability, albeit with a normal sleep pattern. We have investigated the function of FBXL3 in the zebrafish, an excellent model to study both vertebrate development and circadian clock function and, like humans, a diurnal species. Loss of fbxl3a function in zebrafish led to disruption of circadian rhythms of promoter activity and mRNA expression as well as locomotor activity and sleep–wake cycles. However, unlike humans, no morphological effects were evident. These findings point to an evolutionary conserved role for FBXL3 in the circadian clock system across vertebrates and to the acquisition of developmental roles in humans.     

Macrophage-Mediated Melanoma Reduction after HP-NAP Treatment in a Zebrafish Xenograft Model

The Helicobacter pylori Neutrophil Activating Protein (HP-NAP) is endowed with immunomodulatory properties that make it a potential candidate for anticancer therapeutic applications. By activating cytotoxic Th1 responses, HP-NAP inhibits the growth of bladder cancer and enhances the anti-tumor activity of oncolytic viruses in the treatment of metastatic breast cancer and neuroendocrine tumors. The possibility that HP-NAP exerts its anti-tumor effect also by modulating the activity of innate immune cells has not yet been explored. Taking advantage of the zebrafish model, we examined the therapeutic efficacy of HP-NAP against metastatic human melanoma, limiting the observational window to 9 days post-fertilization, well before the maturation of the adaptive immunity. Human melanoma cells were xenotransplanted into zebrafish embryos and tracked in the presence or absence of HP-NAP. The behavior and phenotype of macrophages and the impact of their drug-induced depletion were analyzed exploiting macrophage-expressed transgenes. HP-NAP administration efficiently inhibited tumor growth and metastasis and this was accompanied by strong recruitment of macrophages with a pro-inflammatory profile at the tumor site. The depletion of macrophages almost completely abrogated the ability of HP-NAP to counteract tumor growth. Our findings highlight the pivotal role of activated macrophages in counteracting melanoma growth and support the notion that HP-NAP might become a new biological therapeutic agent for the treatment of metastatic melanomas.     

Evaluation of Epigenetic and Radiomodifying Effects during Radiotherapy Treatments in Zebrafish

Radiotherapy is still a long way from personalizing cancer treatment plans, and its effectiveness depends on the radiosensitivity of tumor cells. Indeed, therapies that are efficient and successful for some patients may be relatively ineffective for others. Based on this, radiobiological research is focusing on the ability of some reagents to make cancer cells more responsive to ionizing radiation, as well as to protect the surrounding healthy tissues from possible side effects. In this scenario, zebrafish emerged as an effective model system to test for radiation modifiers that can potentially be used for radiotherapeutic purposes in humans. The adoption of this experimental organism is fully justified and supported by the high similarity between fish and humans in both their genome sequences and the effects provoked in them by ionizing radiation. This review aims to provide the literature state of the art of zebrafish in vivo model for radiobiological studies, particularly focusing on the epigenetic and radiomodifying effects produced during fish embryos’ and larvae’s exposure to radiotherapy treatments.     

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.     

Comparative Antiseizure Analysis of Diverse Natural Coumarin Derivatives in Zebrafish

Coumarins are a well-known group of plant secondary metabolites with various pharmacological activities, including antiseizure activity. In the search for new antiseizure drugs (ASDs) to treat epilepsy, it is yet unclear which types of coumarins are particularly interesting as a systematic analysis has not been reported. The current study performed behavioral antiseizure activity screening of 18 different coumarin derivatives in the larval zebrafish pentylenetetrazole (PTZ) model using locomotor measurements. Activity was confirmed for seven compounds, which lowered seizure-like behavior as follows: oxypeucedanin 38%, oxypeucedanin hydrate 74%, notopterol 54%, nodakenetin 29%, hyuganin C 35%, daphnoretin 65%, and pimpinellin 60%. These coumarins, together with nodakenin, underwent further antiepileptiform analysis by local field potential recordings from the zebrafish opticum tectum (midbrain). All of them, except for nodakenetin, showed pronounced antiepileptiform activity, decreasing PTZ-induced elevation in power spectral density (PSD) by 83–89% for oxypeucedanin, oxypeucedanin hydrate, and notopterol, 77% for nodakenin, 26% for nodakenetin, 65% for hyuganin C, 88% for daphnoretin, and 81% for pimpinellin. These data demonstrate the potential of diverse coumarin scaffolds for ASD discovery. Finally, the structural differences between active and inactive coumarins were investigated in silico for oxypeucedanin hydrate and byacangelicin for their interaction with GABA-transaminase, a hypothetical target.     

Optogenetic Manipulation of Olfactory Responses in Transgenic Zebrafish: A Neurobiological and Behavioral Study

Olfaction is an important neural system for survival and fundamental behaviors such as predator avoidance, food finding, memory formation, reproduction, and social communication. However, the neural circuits and pathways associated with the olfactory system in various behaviors are not fully understood. Recent advances in optogenetics, high-resolution in vivo imaging, and reconstructions of neuronal circuits have created new opportunities to understand such neural circuits. Here, we generated a transgenic zebrafish to manipulate olfactory signal optically, expressing the Channelrhodopsin (ChR2) under the control of the olfactory specific promoter, omp. We observed light-induced neuronal activity of olfactory system in the transgenic fish by examining c-fos expression, and a calcium indicator suggesting that blue light stimulation caused activation of olfactory neurons in a non-invasive manner. To examine whether the photo-activation of olfactory sensory neurons affect behavior of zebrafish larvae, we devised a behavioral choice paradigm and tested how zebrafish larvae choose between two conflicting sensory cues, an aversive odor or the naturally preferred phototaxis. We found that when the conflicting cues (the preferred light and aversive odor) were presented together simultaneously, zebrafish larvae swam away from the aversive odor. However, the transgenic fish with photo-activation were insensitive to the aversive odor and exhibited olfactory desensitization upon optical stimulation of ChR2. These results show that an aversive olfactory stimulus can override phototaxis, and that olfaction is important in decision making in zebrafish. This new transgenic model will be useful for the analysis of olfaction related behaviors and for the dissection of underlying neural circuits.     

Soybean Meal-Dependent Acute Intestinal Inflammation Delays Osteogenesis in Zebrafish Larvae

Foods are known to be modulators of inflammation and skeletal development. The intestine plays an essential role in the regulation of bone health mainly through the regulation of the absorption of vitamin D and calcium; in fact, inflammatory bowel diseases are often related to bone health issues such as low bone mineral density, high fracture risk, osteoporosis and osteopenia. Considering the complexity of the pathways involved, the use of a simple animal model can be highly useful to better elucidate the pathogenic mechanisms. Soybean flour with a high saponin content has been used in many studies to induce intestinal inflammation in zebrafish larvae. Using a 50% soybean meal (SBM), we analyzed the effects of this soy-induced inflammatory bowel disease on zebrafish larval osteogenesis. Soybean meal induces intestinal functional alterations and an inflammatory state, highlighted by neutral red staining, without altering the general development of the larvae. Our data show that the chondrogenesis as well as endochondral ossification of the head of zebrafish larvae are not affected by an SBM-diet, whereas intramembranous ossification was delayed both in the head, where the length of the ethmoid plate reduced by 17%, and in the trunk with a delayed vertebral mineralization of 47% of SBM larvae. These data highlight that diet-dependent bowel inflammation can differently modulate the different mechanisms of bone development in different zones of the skeleton of zebrafish larvae.     

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.     

Generation of a Triadin KnockOut Syndrome Zebrafish Model

Different forms of sudden cardiac death have been described, including a recently identified form of genetic arrhythmogenic disorder, named “Triadin KnockOut Syndrome” (TKOS). TKOS is associated with recessive mutations in the TRDN gene, encoding for TRIADIN, but the pathogenic mechanism underlying the malignant phenotype has yet to be completely defined. Moreover, patients with TKOS are often refractory to conventional treatment, substantiating the need to identify new therapeutic strategies in order to prevent or treat cardiac events. The zebrafish (Danio rerio) heart is highly comparable to the human heart in terms of functions, signal pathways and ion channels, representing a good model to study cardiac disorders. In this work, we generated the first zebrafish model for trdn loss-of-function, by means of trdn morpholino injections, and characterized its phenotype. Although we did not observe any gross cardiac morphological defect between trdn loss-of-function embryos and controls, we found altered cardiac rhythm that was recovered by the administration of arrhythmic drugs. Our model will provide a suitable platform to study the effect of TRDN mutations and to perform drug screening to identify new pharmacological strategies for patients carrying TRDN mutations.     

Synthesis of New AIEE-Active Chalcones for Imaging of Mitochondria in Living Cells and Zebrafish In Vivo

Fluorophores with aggregation-induced emission enhancement (AIEE) properties have attracted increasing interest in recent years. On the basis of our previous research, we successfully designed and synthesized eleven chalcones. Through an optical performance experiment, we confirmed that compounds 1–6 had obvious AIEE properties. As these AIEE molecules had excellent fluorescence properties and a large Stokes shift, we studied their application in living cell imaging, and the results showed that these compounds had low cytotoxicity and good biocompatibility at the experimental concentrations. More importantly, they could specifically label mitochondria. Subsequently, we selected zebrafish as experimental animals to explore the possibilities of these compounds in animal imaging. The fluorescence imaging of zebrafish showed that these AIEE molecules can enter the embryo and can be targeted to aggregate in the digestive tract, which provides a strong foundation for their practical application in the field of biological imaging. Compared with traditional fluorophores, these AIEE molecules have the advantages of possessing a small molecular weight and high flexibility. Therefore, they have excellent application prospects in the field of biological imaging. In addition, the findings of this study have very positive practical significance for the discovery of more AIEE molecules.