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.     

Ecotoxicology of carbon-based engineered nanoparticles: Effects of fullerene (C60) on aquatic organisms

To more fully assess the toxicity of water-soluble fullerene (nC₆₀), acute toxicity assays were performed on several environmentally relevant species. Included were the freshwater crustaceans Daphnia magna and Hyalella azteca, and a marine harpacticoid copepod, and two fish species, fathead minnow Pimephales promelas and Japanese medaka Oryzias latipes. The latter two species were used to assess sublethal effects of fullerene exposure by also assessing mRNA and protein expression in liver. Because prior studies found that both sonication and using tetrahydrofuran to solubilize fullerene increased the toxicity of nC₆₀, the nC₆₀ used in this study was prepared by stirring. For the invertebrate studies, nC₆₀ could not be prepared at high enough concentration levels to cause 50% mortality (LC₅₀) at 48 or 96 h. The maximum concentrations tested were 35 ppm for freshwater and 22.5 ppm for full-strength (35 ppt) seawater, since at higher concentrations the nC₆₀ precipitated out of solution. Daphnia 21-day exposures resulted in a significant delay in molting and significantly reduced offspring production at 2.5 and 5 ppm nC₆₀, which could possibly produce impacts at the population-level. For the fish, we found that neither the mRNA nor protein-expression levels of cytochrome P450 isozymes CYP1A, CYP2K1 and CYP2M1 were changed. The peroxisomal lipid transport protein PMP70 was significantly reduced in fathead minnow, but not medaka, indicating potential changes in acyl-CoA pathways.     

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.     

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.     

Tryptophan Hydroxylase 2 Deficiency Modifies the Effects of Fluoxetine and Pargyline on the Behavior, 5-HT- and BDNF-Systems in the Brain of Zebrafish (Danio rerio)

The mechanisms of resistance to antidepressant drugs is a key and still unresolved problem of psychopharmacology. Serotonin (5-HT) and brain-derived neurotrophic factor (BDNF) play a key role in the therapeutic effect of many antidepressants. Tryptophan hydroxylase 2 (TPH2) is the rate-limiting enzyme in 5-HT synthesis in the brain. We used zebrafish (Danio rerio) as a promising model organism in order to elucidate the effect of TPH2 deficiency caused by p-chlorophenylalanine (pCPA) on the alterations in behavior and expression of 5-HT-related (Tph2, Slc6a4b, Mao, Htr1aa, Htr2aa) and BDNF-related (Creb, Bdnf, Ntrk2a, Ngfra) genes in the brain after prolonged treatment with two antidepressants, inhibitors of 5-HT reuptake (fluoxetine) and oxidation (pargyline). In one experiment, zebrafish were treated for 72 h with 0.2 mg/L fluoxetine, 2 mg/L pCPA, or the drugs combination. In another experiment, zebrafish were treated for 72 h with 0.5 mg/L pargyline, 2 mg/L pCPA, or the drugs combination. Behavior was studied in the novel tank diving test, mRNA levels were assayed by qPCR, 5-HT and its metabolite concentrations were measured by HPLC. The effects of interaction between pCPA and the drugs on zebrafish behavior were observed: pCPA attenuated “surface dwelling” induced by the drugs. Fluoxetine decreased mRNA levels of Tph2 and Htr2aa genes, while pargyline decreased mRNA levels of Slc6a4b and Htr1aa genes. Pargyline reduced Creb, Bdnf and Ntrk2a genes mRNA concentration only in the zebrafish treated with pCPA. The results show that the disruption of the TPH2 function can cause a refractory to antidepressant treatment.     

Cytogenomics Unveil Possible Transposable Elements Driving Rearrangements in Chromosomes 2 and 4 of Solea senegalensis

Cytogenomics, the integration of cytogenetic and genomic data, has been used here to reconstruct the evolution of chromosomes 2 and 4 of Solea senegalensis. S. senegalensis is a flat fish with a karyotype comprising 2n = 42 chromosomes: 6 metacentric + 4 submetacentric + 8 subtelocentric + 24 telocentric. The Fluorescence in situ Hybridization with Bacterial Artificial Chromosomes (FISH-BAC) technique was applied to locate BACs in these chromosomes (11 and 10 BACs in chromosomes 2 and 4, respectively) and to generate integrated maps. Synteny analysis, taking eight reference fish species (Cynoglossus semilaevis, Scophthalmus maximus, Sparus aurata, Gasterosteus aculeatus, Xiphophorus maculatus, Oryzias latipes, Danio rerio, and Lepisosteus oculatus) for comparison, showed that the BACs of these two chromosomes of S. senegalensis were mainly distributed in two principal chromosomes in the reference species. Transposable Elements (TE) analysis showed significant differences between the two chromosomes, in terms of number of loci per Mb and coverage, and the class of TE (I or II) present. Analysis of TE divergence in chromosomes 2 and 4 compared to their syntenic regions in four reference fish species (C. semilaevis, S. maximus, O. latipes, and D. rerio) revealed differences in their age of activity compared with those species but less notable differences between the two chromosomes. Differences were also observed in peaks of divergence and coverage of TE families for all reference species even in those close to S. senegalensis, like S. maximus and C. semilaevis. Considered together, chromosomes 2 and 4 have evolved by Robertsonian fusions, pericentric inversions, and other chromosomal rearrangements mediated by TEs.     

Proteome Analysis of Whole-Body Responses in Medaka Experimentally Exposed to Fish-Killing Dinoflagellate Karenia mikimotoi

Karenia mikimotoi is a well-known harmful algal bloom species. Blooms of this dinoflagellate have become a serious threat to marine life, including fish, shellfish, and zooplanktons and are usually associated with massive fish death. Despite the discovery of several toxins such as gymnocins and gymnodimines in K. mikimotoi, the mechanisms underlying the ichthyotoxicity of this species remain unclear, and molecular studies on this topic have never been reported. The present study investigates the fish-killing mechanisms of K. mikimotoi through comparative proteomic analysis. Marine medaka, a model fish organism, was exposed to K. mikimotoi for a three-part time period (LT₂₅, LT₅₀ and LT₉₀). Proteins extracted from the whole fish were separated by using two-dimensional gel electrophoresis, and differentially expressed proteins were identified with reference to an untreated control. The change in fish proteomes over the time-course of exposure were analyzed. A total of 35 differential protein spots covering 19 different proteins were identified, of which most began to show significant change in expression levels at the earliest stage of intoxication. Among the 19 identified proteins, some are closely related to the oxidative stress responses, energy metabolism, and muscle contraction. We propose that oxidative stress-mediated muscle damage might explain the symptoms developed during the ichthyotoxicity test, such as gasping for breath, loss of balance, and body twitching. Our findings lay the foundations for more in-depth studies of the mechanisms of K. mikimotoi’s ichthyotoxicity.     

Molecular Evolution of Aralkylamine N-Acetyltransferase in Fish: A Genomic Survey

All living organisms synchronize biological functions with environmental changes; melatonin plays a vital role in regulating daily and seasonal variations. Due to rhythmic activity of the timezyme aralkylamine N-acetyltransferase (AANAT), the blood level of melatonin increases at night and decreases during daytime. Whereas other vertebrates have a single form of AANAT, bony fishes possess various isoforms of aanat genes, though the reasons are still unclear. Here, we have taken advantage of multiple unpublished teleost aanat sequences to explore and expand our understanding of the molecular evolution of aanat in fish. Our results confirm that two rounds of whole-genome duplication (WGD) led to the existence of three fish isoforms of aanat, i.e., aanat1a, aanat1b, and aanat2; in addition, gene loss led to the absence of some forms from certain special fish species. Furthermore, we suggest the different roles of two aanat1s in amphibious mudskippers, and speculate that the loss of aanat1a, may be related to terrestrial vision change. Several important sites of AANAT proteins and regulatory elements of aanat genes were analyzed for structural comparison and functional forecasting, respectively, which provides insights into the molecular evolution of the differences between AANAT1 and AANAT2.     

Acute Toxicity of Cu-MOF Nanoparticles (nanoHKUST-1) towards Embryos and Adult Zebrafish

Metal-organic frameworks (MOFs) demonstrate unique properties, which are prospective for drug delivery, catalysis, and gas separation, but their biomedical applications might be limited due to their obscure interactions with the environment and humans. It is important to understand their toxic effect on nature before their wide practical application. In this study, HKUST-1 nanoparticles (Cu-nanoMOF, Cu₃(btc)₂, btc = benzene-1,3,5-tricarboxylate) were synthesized by the microwave (MW)-assisted ionothermal method and characterized by X-ray powder diffraction (XRD) and transmission electron microscopy (TEM) techniques. The embryotoxicity and acute toxicity of HKUST-1 towards embryos and adult zebrafish were investigated. To gain a better understanding of the effects of Cu-MOF particles towards Danio rerio (D. rerio) embryos were exposed to HKUST-1 nanoparticles (NPs) and Cu²⁺ ions (CuSO₄). Cu²⁺ ions showed a higher toxic effect towards fish compared with Cu-MOF NPs for D. rerio. Both forms of fish were sensitive to the presence of HKUST-1 NPs. Estimated LC₅₀ values were 2.132 mg/L and 1.500 mg/L for zebrafish embryos and adults, respectively. During 96 h of exposure, the release of copper ions in a stock solution and accumulation of copper after 96 h were measured in the internal organs of adult fishes. Uptake examination of the major internal organs did not show any concentration dependency. An increase in the number of copper ions in the test medium was found on the first day of exposure. Toxicity was largely restricted to copper release from HKUST-1 nanomaterials structure into solution.     

Evolution of MicroRNA Biogenesis Genes in the Sterlet (Acipenser ruthenus) and Other Polyploid Vertebrates

MicroRNAs play a crucial role in eukaryotic gene regulation. For a long time, only little was known about microRNA-based gene regulatory mechanisms in polyploid animal genomes due to difficulties of polyploid genome assembly. However, in recent years, several polyploid genomes of fish, amphibian, and even invertebrate species have been sequenced and assembled. Here we investigated several key microRNA-associated genes in the recently sequenced sterlet (Acipenser ruthenus) genome, whose lineage has undergone a whole genome duplication around 180 MYA. We show that two paralogs of drosha, dgcr8, xpo1, and xpo5 as well as most ago genes have been retained after the acipenserid-specific whole genome duplication, while ago1 and ago3 genes have lost one paralog. While most diploid vertebrates possess only a single copy of dicer1, we strikingly found four paralogs of this gene in the sterlet genome, derived from a tandem segmental duplication that occurred prior to the last whole genome duplication. ago1,3,4 and exportins1,5 look to be prone to additional segment duplications producing up to four-five paralog copies in ray-finned fishes. We demonstrate for the first time exon microsatellite amplification in the acipenserid drosha2 gene, resulting in a highly variable protein product, which may indicate sub- or neofunctionalization. Paralogous copies of most microRNA metabolism genes exhibit different expression profiles in various tissues and remain functional despite the rediploidization process. Subfunctionalization of microRNA processing gene paralogs may be beneficial for different pathways of microRNA metabolism. Genetic variability of microRNA processing genes may represent a substrate for natural selection, and, by increasing genetic plasticity, could facilitate adaptations to changing environments.     

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.     

Dimethylsulfoniopropionate (DMSP) Increases Survival of Larval Sablefish, <Emphasis Type="Italic">Anoplopoma fimbria</Emphasis>

High concentrations of dimethylsulfoniopropionate (DMSP), a chemical compound released by lysed phytoplankton, may indicate high rates of grazing by zooplankton and may thus be a foraging cue for planktivorous fishes. Previous studies have shown that some planktivorous fishes and birds aggregate or alter locomotory behavior in response to this chemical cue, which is likely adaptive because it helps them locate prey. These behavioral responses have been demonstrated in juveniles and adults, but no studies have tested for effects on larval fish. Larvae suffer from high mortality rates and are vulnerable to starvation. While larvae are generally thought to be visual predators, they actually have poor vision and cryptic prey. Thus, larval fish should benefit from a chemical cue that provides information on prey abundance. We reared larval sablefish, Anoplopoma fimbria, for one week and supplemented feedings with varying concentrations of DMSP to test the hypothesis that DMSP affects larval survival. Ecologically relevant DMSP concentrations increased larval survival by up to 70 %, which has implications for production in aquaculture and recruitment in nature. These results provide a new tool for increasing larval production in aquaculture and also suggest that larvae may use DMSP as an olfactory cue. The release of DMSP may be a previously unappreciated mechanism through which phytoplankton affect larval survival and recruitment.     

Detection and Modulation of Olfactory Sensing Receptors in Carnivorous Rainbow Trout (Oncorhynchus mykiss) Fed from First Feeding with Plant-Based Diet

Sense of smell is mediated by diverse families of olfactory sensing receptors, conveying important dietary information, fundamental for growth and survival. The aim of this study was to elucidate the role of the sensory olfactory pathways in the regulation of feeding behavior of carnivorous rainbow trout (RT, Oncorhynchus mykiss), from first feeding until 8 months. Compared to a commercial diet, RT fed with a total plant-based diet showed drastically altered growth performance associated with feed intake from an early stage. Exhaustive examination of an RT genome database identified three vomeronasal type 1 receptor-like (ORA), 10 vomeronasal type 2 receptor-like (OLFC) and 14 main olfactory receptor (MOR) genes, all highly expressed in sensory organs, indicating their potential functionality. Gene expression after feeding demonstrated the importance in olfactory sensing perception of some OLFC (olfcg6) and MOR (mor103, -107, -112, -113, -133) receptor family genes in RT. The gene ora1a showed evidence of involvement in olfactory sensing perception for fish fed with a commercial-like diet, while ora5b, mor118, mor124 and olfch1 showed evidence of involvement in fish fed with a plant-based diet. Results indicated an impact of a plant-based diet on the regulation of olfactory sensing pathways as well as influence on monoaminergic neurotransmission in brain areas related to olfactory-driven behaviors. The overall findings suggest that feeding behavior is mediated through olfactory sensing detection and olfactory-driven behavior pathways in RT.     

Zebrafish Bioassay for Screening Therapeutic Candidates Based on Melanotrophic Activity

In this study, we used the zebrafish animal model to establish a bioassay by which physiological efficacy differential of alpha-melanocyte-stimulating hormone (α-MSH) analogues could be measured by melanosome dispersion in zebrafish larvae. Brain-skin connection research has purported the interconnectedness between the nervous system and skin physiology. Accordingly, the neuropeptide α-MSH is a key regulator in several physiological processes, such as skin pigmentation in fish. In mammals, α-MSH has been found to regulate motivated behavior, appetite, and emotion, including stimulation of satiety and anxiety. Several clinical and animal model studies of autism spectrum disorder (ASD) have already demonstrated the effectiveness of α-MSH in restoring the social deficits of autism. Therefore, we sought to analyze the effect of synthetic and naturally-occurring α-MSH variants amongst different species. Our results showed that unique α-MSH derivatives from several fish species produced differential effects on the degree of melanophore dispersion. Using α-MSH human form as a standard, we could identify derivatives that induced greater physiological effects; particularly, the synthetic analogue melanotan-II (MT-II) exhibited a higher capacity for melanophore dispersion than human α-MSH. This was consistent with previous findings in an ASD mouse model demonstrating the effectiveness of MT-II in improving ASD behavioral symptoms. Thus, the melanophore assay may serve as a useful screening tool for therapeutic candidates for novel drug discovery.     

A new model for water adsorption in porous ceramics

This research proposes a new isotherm model that is derived for adsorption of clustering and condensable vapors on mesoporous ceramics. The adsorption characteristics of water vapor on both experimental data and isotherm model were investigated over the relative pressure ranging from 0 to 0.95. The applicability of the model was evaluated by significance of fitting parameters. For each adsorption isotherm there are five parameters (m, q _f , K _f , q _m and K _m ). The comparison of modeling fit with experimental data was used as the methodology for selecting the most informative and the best-fitting model. The experimental testing results showed that this model is able to describe all possible behaviors of the water adsorption isotherm displaying type IV classification. The model is based on the forming and growth of the water molecule clusters and capillary condensation mechanism in mesopore. The findings suggest a significant tool for understanding the behavior of water adsorption in humidity-control porous ceramics.     

PCD Genes—From Patients to Model Organisms and Back to Humans

Primary ciliary dyskinesia (PCD) is a hereditary genetic disorder caused by the lack of motile cilia or the assembxly of dysfunctional ones. This rare human disease affects 1 out of 10,000–20,000 individuals and is caused by mutations in at least 50 genes. The past twenty years brought significant progress in the identification of PCD-causative genes and in our understanding of the connections between causative mutations and ciliary defects observed in affected individuals. These scientific advances have been achieved, among others, due to the extensive motile cilia-related research conducted using several model organisms, ranging from protists to mammals. These are unicellular organisms such as the green alga Chlamydomonas, the parasitic protist Trypanosoma, and free-living ciliates, Tetrahymena and Paramecium, the invertebrate Schmidtea, and vertebrates such as zebrafish, Xenopus, and mouse. Establishing such evolutionarily distant experimental models with different levels of cell or body complexity was possible because both basic motile cilia ultrastructure and protein composition are highly conserved throughout evolution. Here, we characterize model organisms commonly used to study PCD-related genes, highlight their pros and cons, and summarize experimental data collected using these models.