Zebrafish models of Tauopathy

Tauopathies are a group of incurable neurodegenerative diseases, in which loss of neurons is accompanied by intracellular deposition of fibrillar material composed of hyperphosphorylated forms of the microtubule-associated protein Tau. A zebrafish model of Tauopathy could complement existing murine models by providing a platform for genetic and chemical screens, in order to identify novel therapeutic targets and compounds with disease-modifying potential. In addition, Tauopathy zebrafish would be useful for hypothesis-driven experiments, especially those exploiting the potential to deploy in vivo imaging modalities. Several considerations, including conservation of specialized neuronal and other cellular populations, and biochemical pathways implicated in disease pathogenesis, suggest that the zebrafish brain is an appropriate setting in which to model these complex disorders. Novel transgenic zebrafish lines expressing wild-type and mutant forms of human Tau in CNS neurons have recently been reported. These studies show evidence that human Tau undergoes disease-relevant changes in zebrafish neurons, including somato-dendritic relocalization, hyperphosphorylation and aggregation. In addition, preliminary evidence suggests that Tau transgene expression can precipitate neuronal dysfunction and death. These initial studies are encouraging that the zebrafish holds considerable promise as a model in which to study Tauopathies. Further studies are necessary to clarify the phenotypes of transgenic lines and to develop assays and models suitable for unbiased high-throughput screening approaches. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.     

The Study of Glioma by Xenotransplantation in Zebrafish Early Life Stages

Zebrafish (Danio rerio) and their transparent embryos are becoming an increasingly popular tool for studying processes involved in tumor progression and in the search for novel tumor treatment approaches. The xenotransplantation of fluorescently labeled mammalian cancer cells into zebrafish embryos is an approach enabling relatively high-throughput in vivo analyses. The small size of the embryos as well as the relative simplicity of their manipulation and maintenance allow for large numbers of embryos to be processed efficiently in a short time and at low cost. Furthermore, the possibility of fluorescence microscopic imaging of tumor progression within zebrafish embryos and larvae holds unprecedented potential for the real-time visualization of these processes in vivo. This review presents the methodologies of xenotransplantation studies on zebrafish involving research on tumor invasion, proliferation, tumor-induced angiogenesis and screening for antitumor therapeutics. We further focus on the application of these zebrafish to the study of glioma; in particular, its most common and malignant form, glioblastoma.     

Perspectives on genetic animal models of serotonin toxicity

Serotonin syndrome, or serotonin toxicity, is a serious disorder attributable to exaggerated serotonergic function in the brain, most commonly after antidepressant overdose or after combining several psychotropic medications. Similar condition (serotonin syndrome-like behavior) can be evoked in animals experimentally, following administration of serotonergic drugs. In addition to pharmacological stimulation, some genetic and other factors may contribute to serotonin toxicity, prompting the need for new experimental genetic models relevant to this disorder. Here we discuss current problems and perspectives regarding genetic animal models of serotonin-related syndromes, and outline the potential utility of these models in experimental neurochemistry and clinical research.     

Influenza A virus infection in zebrafish recapitulates mammalian infection and sensitivity to anti-influenza drug treatment

Seasonal influenza virus infections cause annual epidemics and sporadic pandemics. These present a global health concern, resulting in substantial morbidity, mortality and economic burdens. Prevention and treatment of influenza illness is difficult due to the high mutation rate of the virus, the emergence of new virus strains and increasing antiviral resistance. Animal models of influenza infection are crucial to our gaining a better understanding of the pathogenesis of and host response to influenza infection, and for screening antiviral compounds. However, the current animal models used for influenza research are not amenable to visualization of host-pathogen interactions or high-throughput drug screening. The zebrafish is widely recognized as a valuable model system for infectious disease research and therapeutic drug testing. Here, we describe a zebrafish model for human influenza A virus (IAV) infection and show that zebrafish embryos are susceptible to challenge with both influenza A strains APR8 and X-31 (Aichi). Influenza-infected zebrafish show an increase in viral burden and mortality over time. The expression of innate antiviral genes, the gross pathology and the histopathology in infected zebrafish recapitulate clinical symptoms of influenza infections in humans. This is the first time that zebrafish embryos have been infected with a fluorescent IAV in order to visualize infection in a live vertebrate host, revealing a pattern of vascular endothelial infection. Treatment of infected zebrafish with a known anti-influenza compound, Zanamivir, reduced mortality and the expression of a fluorescent viral gene product, demonstrating the validity of this model to screen for potential antiviral drugs. The zebrafish model system has provided invaluable insights into host-pathogen interactions for a range of infectious diseases. Here, we demonstrate a novel use of this species for IAV research. This model has great potential to advance our understanding of influenza infection and the associated host innate immune response.KEY WORDS: Influenza, Zebrafish, Virus, Innate immunity     

Artesunate inhibits Sjögren's syndrome-like autoimmune responses and BAFF-induced B cell hyperactivation via TRAF6-mediated NF-κB signaling

BackgroundHyperactivation of B cells by activators has been demonstrated to play a central role in the pathogenesis of Sjögren's syndrome (SS). In this study, we found that artesunate (ART) can attenuate BAFF-induced B cell hyperactivation and SS-like symptoms in NOD/Ltj mice.PurposeTo determine the efficacy of ART in attenuating SS-like symptoms in vivo and explore the underlying mechanism in vitro.Study designART was intragastrically injected into SS-like NOD/Ltj mice. The cytokine hsBAFF was used to activate Raji and Daudi B cells to mimic B cell hyperactivation in vitro.MethodsThe efficacy of ART in inhibiting SS progression was studied in NOD/Ltj mice. Salivary flow rate, the number of lymphocytic infiltration foci, the level of autoantibodies and the extent of B cell infiltration were measured in the indicated groups. CCK-8 assays, flow cytometry-based EdU staining and Annexin V/PI staining were also used to detect the effect of ART on the survival and proliferation mechanism in BAFF-induced Raji and Daudi cells. Further studies determined that TRAF6 degradation is a potential mechanism by which ART determines B cell fate.ResultsTreatment with ART inhibited lymphocytic foci formation, B cell infiltration and autoantibody secretion in SS-like NOD/Ltj mice. In vitro assay results indicated that ART effectively inhibited BAFF-induced viability, survival and proliferation of neoplastic B cells. Mechanistically, ART targeted BAFF-activated NFκB by regulating the proteasome-mediated degradation of TRAF6 in Raji and Daudi cells.ConclusionART ameliorated murine SS-like symptoms and regulated TRAF6-NFκB signaling, thus determining survival and proliferation of B cells.     

Zebrafish Tyrosine Hydroxylase 2 Gene Encodes Tryptophan Hydroxylase

The primary pathological hallmark of Parkinson disease (PD) is the profound loss of dopaminergic neurons in the substantia nigra pars compacta. To facilitate the understanding of the underling mechanism of PD, several zebrafish PD models have been generated to recapitulate the characteristics of dopaminergic (DA) neuron loss. In zebrafish studies, tyrosine hydroxylase 1 (th1) has been frequently used as a molecular marker of DA neurons. However, th1 also labels norepinephrine and epinephrine neurons. Recently, a homologue of th1, named tyrosine hydroxylase 2 (th2), was identified based on the sequence homology and subsequently used as a novel marker of DA neurons. In this study, we present evidence that th2 co-localizes with serotonin in the ventral diencephalon and caudal hypothalamus in zebrafish embryos. In addition, knockdown of th2 reduces the level of serotonin in the corresponding th2-positive neurons. This phenotype can be rescued by both zebrafish th2 and mouse tryptophan hydroxylase 1 (Tph1) mRNA as well as by 5-hydroxytryptophan, the product of tryptophan hydroxylase. Moreover, the purified Th2 protein has tryptophan hydroxylase activity comparable with that of the mouse TPH1 protein in vitro. Based on these in vivo and in vitro results, we conclude that th2 is a gene encoding for tryptophan hydroxylase and should be used as a marker gene of serotonergic neurons.     

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system^1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 μl during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds⁴. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish^5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.     

Sj?gren's syndrome: studying the disease in mice

Sj?gren's syndrome (SS), a systemic autoimmune disease, is characterized by inflammation of exocrine tissues accompanied by a significant loss of their secretory function. Clinical symptoms develop late and there are no diagnostic tests enabling early diagnosis of SS. Thus, particularly to study these covert stages, researchers turn to studying animal models where mice provide great freedom for genetic manipulation and testing the effect of experimental intervention. The present review summarizes current literature pertaining to both spontaneous and extrinsic-factor induced SS-like diseases in mouse models, discussing advantages and disadvantages related to the use of murine models in SS research.     

High-throughput screening identified mitoxantrone to induce death of hepatocellular carcinoma cells with autophagy involvement

The use of highly efficient high-throughput screening (HTS) platform has recently gained more attention as a plausible approach to identify de novo therapeutic application potential of conventional anti-tumor drugs for cancer treatments. In this study, we used hepatocellular carcinoma (HCC) cells as models to identify cytotoxic compounds by HTS. To identify cytotoxic compounds for potential HCC treatments, 3271 compounds from three well established small molecule libraries were screened against HCC cell lines. Thirty-two small molecules were identified from the primary screen to induce cell death. Particularly, mitoxantrone (MTX), which is an established antineoplastic drug, significantly and specifically inhibited the growth and proliferation of HCC cells in vitro. Mechanistic studies of LC3-II, p62 and phosphorylation of p70S6K in HepG2 cells revealed that MTX treatment induced mTOR-dependent autophagy activation, which was further confirmed by the autophagic flux assay using lysosomal inhibitor chloroquine (CQ). In the combined treatment of MTX and CQ, where autophagy was inhibited by CQ, the elevations of cleaved Caspase-3 and PARP were observed, indicating the enhanced apoptosis in HepG2 cells. Taken together, we hypothesize that MTX-induced autophagy plays an pro-survival role in HCC treatment. Combined treatment with autophagy inhibitor may combat the chemo-resistance of HCC to MTX treatment and therefore deserves future clinical investment.     

Sjögren's syndrome: studying the disease in mice

Sj?gren's syndrome (SS), a systemic autoimmune disease, is characterized by inflammation of exocrine tissues accompanied by a significant loss of their secretory function. Clinical symptoms develop late and there are no diagnostic tests enabling early diagnosis of SS. Thus, particularly to study these covert stages, researchers turn to studying animal models where mice provide great freedom for genetic manipulation and testing the effect of experimental intervention. The present review summarizes current literature pertaining to both spontaneous and extrinsic-factor induced SS-like diseases in mouse models, discussing advantages and disadvantages related to the use of murine models in SS research.     

A Novel Zebrafish Xenotransplantation Model for Study of Glioma Stem Cell Invasion

Invasion and metastasis of solid tumors are the major causes of death in cancer patients. Cancer stem cells (CSCs) constitute a small fraction of tumor cell population, but play a critical role in tumor invasion and metastasis. The xenograft of tumor cells in immunodeficient mice is one of commonly used in vivo models to study the invasion and metastasis of cancer cells. However, this model is time-consuming and labor intensive. Zebrafish (Danio rerio) and their transparent embryos are emerging as a promising xenograft tumor model system for studies of tumor invasion. In this study, we established a tumor invasion model by using zebrafish embryo xenografted with human glioblastoma cell line U87 and its derived cancer stem cells (CSCs). We found that CSCs-enriched from U87 cells spreaded via the vessels within zebrafish embryos and such cells displayed an extremely high level of invasiveness which was associated with the up-regulated MMP-9 by CSCs. The invasion of glioma CSCs (GSCs) in zebrafish embryos was markedly inhibited by an MMP-9 inhibitor. Thus, our zebrafish embryo model is considered a cost-effective approach tostudies of the mechanisms underlying the invasion of CSCs and suitable for high-throughput screening of novel anti-tumor invasion/metastasis agents.     

鸡下丘脑内生长抑素样成分的分布

SS (somatostatin) is widely distributed in the bodies of different animals from the low invertabrates (such as protozoa) to vertabrates.The distribution of SS in the brain varies in species.In mammalian brain,except cortex,hypothalamus contains the highest SS level (Zhou,1989).Evidences for SS innervation in the mammalian and human hypothalamus have been obtained by a number of investigators.However,the reports are very few in birds.The present paper disclosed the distribution of SS-like immunoreactivity in the chicken hypothalamus by using immunocytochemical (ABC) technique to provide phylogenetical evidence for a better understanding of SS function.     

Fishing for causes and cures of motor neuron disorders

Motor neuron disorders (MNDs) are a clinically heterogeneous group of neurological diseases characterized by progressive degeneration of motor neurons, and share some common pathological pathways. Despite remarkable advances in our understanding of these diseases, no curative treatment for MNDs exists. To better understand the pathogenesis of MNDs and to help develop new treatments, the establishment of animal models that can be studied efficiently and thoroughly is paramount. The zebrafish (Danio rerio) is increasingly becoming a valuable model for studying human diseases and in screening for potential therapeutics. In this Review, we highlight recent progress in using zebrafish to study the pathology of the most common MNDs: spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia (HSP). These studies indicate the power of zebrafish as a model to study the consequences of disease-related genes, because zebrafish homologues of human genes have conserved functions with respect to the aetiology of MNDs. Zebrafish also complement other animal models for the study of pathological mechanisms of MNDs and are particularly advantageous for the screening of compounds with therapeutic potential. We present an overview of their potential usefulness in MND drug discovery, which is just beginning and holds much promise for future therapeutic development.KEY WORDS: ALS, HSP, SMA, Zebrafish, Drug discovery, Motor neuron disorders     

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.     

Significance of the zebrafish model in the discovery of bioactive molecules from nature

Natural products have immense therapeutic potential not only due to their structural variation and complexity but also due to their range of biological activities. Research based on natural products has led to the discovery of molecules with biomedical and pharmaceutical applications in different therapeutic areas like cancer, inflammation responses, diabetes, and infectious diseases. There are still several challenges to be overcome in natural product drug discovery research programs and the challenge of high throughput screening of natural substances is one of them. Bioactivity screening is an integral part of the drug discovery process and several in vitro and in vivo biological models are now available for this purpose. Among other well-reported biological models, the zebrafish (Danio rerio) is emerging as an important in vivo model for preclinical studies of synthetic molecules in different therapeutic areas. Zebrafish embryos have a short reproductive cycle, show ease of maintenance at high densities in the laboratory and administration of drugs is a straightforward procedure. The embryos are optically transparent, allowing for the visualization of drug effects on internal organs during the embryogenesis process. In this review, we illustrate the importance of using zebrafish as an important biological model in the discovery of bioactive drugs from natural sources.     

Three-dimensional neurophenotyping of adult zebrafish behavior

The use of adult zebrafish (Danio rerio) in neurobehavioral research is rapidly expanding. The present large-scale study applied the newest video-tracking and data-mining technologies to further examine zebrafish anxiety-like phenotypes. Here, we generated temporal and spatial three-dimensional (3D) reconstructions of zebrafish locomotion, globally assessed behavioral profiles evoked by several anxiogenic and anxiolytic manipulations, mapped individual endpoints to 3D reconstructions, and performed cluster analysis to reconfirm behavioral correlates of high- and low-anxiety states. The application of 3D swim path reconstructions consolidates behavioral data (while increasing data density) and provides a novel way to examine and represent zebrafish behavior. It also enables rapid optimization of video tracking settings to improve quantification of automated parameters, and suggests that spatiotemporal organization of zebrafish swimming activity can be affected by various experimental manipulations in a manner predicted by their anxiolytic or anxiogenic nature. Our approach markedly enhances the power of zebrafish behavioral analyses, providing innovative framework for high-throughput 3D phenotyping of adult zebrafish behavior.     

Small molecule screening in the zebrafish

The zebrafish is an ideal organism for small molecule studies. The ability to use the whole organism allows complex in vivo phenotypes to be assayed and combines animal testing with screening. Embryos are easily treatable by waterborne exposure. The small size and abundance of embryos make zebrafish suitable for screening in a high-throughput manner in 96- or 48-well plates. Zebrafish embryos have successfully been used in chemical genetic screens to elucidate biological pathways and find chemical suppressors. Small molecules discovered by screening zebrafish disease models may also be useful as lead compounds for drug development as there appears to be a high level of conservation of drug activity between mammals and zebrafish. Here we provide the technical aspects of treating embryos with small molecules and performing chemical screens with zebrafish.     

Quantitative Phenotyping-Based In Vivo Chemical Screening in a Zebrafish Model of Leukemia Stem Cell Xenotransplantation

Zebrafish-based chemical screening has recently emerged as a rapid and efficient method to identify important compounds that modulate specific biological processes and to test the therapeutic efficacy in disease models, including cancer. In leukemia, the ablation of leukemia stem cells (LSCs) is necessary to permanently eradicate the leukemia cell population. However, because of the very small number of LSCs in leukemia cell populations, their use in xenotransplantation studies (in vivo) and the difficulties in functionally and pathophysiologically replicating clinical conditions in cell culture experiments (in vitro), the progress of drug discovery for LSC inhibitors has been painfully slow. In this study, we developed a novel phenotype-based in vivo screening method using LSCs xenotransplanted into zebrafish. Aldehyde dehydrogenase-positive (ALDH+) cells were purified from chronic myelogenous leukemia K562 cells tagged with a fluorescent protein (Kusabira-orange) and then implanted in young zebrafish at 48 hours post-fertilization. Twenty-four hours after transplantation, the animals were treated with one of eight different therapeutic agents (imatinib, dasatinib, parthenolide, TDZD-8, arsenic trioxide, niclosamide, salinomycin, and thioridazine). Cancer cell proliferation, and cell migration were determined by high-content imaging. Of the eight compounds that were tested, all except imatinib and dasatinib selectively inhibited ALDH+ cell proliferation in zebrafish. In addition, these anti-LSC agents suppressed tumor cell migration in LSC-xenotransplants. Our approach offers a simple, rapid, and reliable in vivo screening system that facilitates the phenotype-driven discovery of drugs effective in suppressing LSCs.     

A zebrafish (danio rerio) model for high-throughput screening food and drugs with uric acid-lowering activity

With co-treatment of potassium oxonate (PO) and xanthine sodium salt (XSS), a zebrafish larva model of acute hyperuricemia has been constructed for the first time. The results show PO 200 μM + XSS 10 μM, PO 300 μM + XSS 15 μM, and PO 400 μM + XSS 20 μM can significantly increase the level of uric acid in the zebrafish larvae (P?<?0.05), the concentrations as described above can be used to construct the zebrafish larvae model of acute hyperuricemia. At the same time, treatment of allopurinol (APL, one of the hyperuricemia drugs) at 2000?μM?(P?<?0.001) and treatment of anserine (ASE) at 200?μM?(P < 0.05) could significantly decrease the level of uric acid in the model group which received PO 200 μM + XSS 10 μM, which demonstrate that such model could offer a new robust approach for high-throughput screening of food and drugs with uric acid-lowering activity.