Zebrafish larvae show a clear and distinct pattern of swimming in response to light and dark conditions, following the development of a swim bladder at 4 days post fertilization. This swimming ...behavior is increasingly employed in the screening of neuroactive drugs. The recent emergence of high-throughput techniques for the automatic tracking of zebrafish larvae has further allowed an objective and efficient way of finding subtle behavioral changes that could go unnoticed during manual observations. This review highlights the use of zebrafish larvae as a high-throughput behavioral model for the screening of neuroactive compounds. We describe, in brief, the behavior repertoire of zebrafish larvae. Then, we focus on the utilization of light-dark locomotion test in identifying and screening of neuroactive compounds.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is spreading worldwide with different clinical manifestations. Age and comorbidities may explain severity in critical cases and ...people living with human immunodeficiency virus (HIV) might be at particularly high risk for severe progression. Nonetheless, current data, although sometimes contradictory, do not confirm higher morbidity, risk of more severe COVID-19 or higher mortality in HIV-infected people with complete access to antiretroviral therapy (ART). A possible protective role of ART has been hypothesized to explain these observations. Anti-viral drugs used to treat HIV infection have been repurposed for COVID-19 treatment; this is also based on previous studies on severe acute respiratory syndrome virus (SARS-CoV) and Middle East respiratory syndrome virus (MERS-CoV). Among them, lopinavir/ritonavir, an inhibitor of viral protease, was extensively used early in the pandemic but it was soon abandoned due to lack of effectiveness in clinical trials. However, remdesivir, a nucleotide analog that acts as reverse-transcriptase inhibitor, which was tested early during the pandemic because of its wide range of antiviral activity against several RNA viruses and its safety profile, is currently the only antiviral medication approved for COVID-19. Tenofovir, another nucleotide analog used extensively for HIV treatment and pre-exposure prophylaxis (PrEP), has also been hypothesized as effective in COVID-19. No data on tenofovir’s efficacy in coronavirus infections other than COVID-19 are currently available, although information relating to SARS-CoV-2 infection is starting to come out. Here, we review the currently available evidence on tenofovir’s efficacy against SARS-CoV-2.
As most new medications, Cabotegravir (CAB) was recently approved as an antiretroviral treatment of HIV infection without in-depth safety information on in utero exposure. Although no developmental ...toxicity in rats and rabbits was reported, recent studies demonstrated that CAB decreases pluripotency of human embryonic stem cells. CAB exposure effects during development were assessed in zebrafish embryos by the Fish Embryo Toxicity test after exposure at subtherapeutic concentrations up to 25× the human C
. Larvae behavior was assessed by the light-dark locomotion test. The expression of factors involved in neurogenesis was evaluated by whole-mount in situ hybridization. CAB did not cause gross morphological defects at low doses, although pericardial edema, uninflated swim bladder, decreased heartbeats, growth delay, and decreased hatching rate were observed at the highest concentrations. Decreased locomotion was observed even at the subtherapeutic dose, suggesting alterations of nervous system integrity. This hypothesis was supported by the observation of decreased expression of crucial factors involved in early neuronal differentiation in diencephalic and telencephalic dopaminergic areas, midbrain/hindbrain boundary, and craniofacial ganglia. These findings support CAB effects on neurogenesis in zebrafish embryos and suggest long-term follow-up of exposed infants to provide data on drug safety during pregnancy.
Dolutegravir (DTG) is one of the most prescribed antiretroviral drugs for treating people with HIV infection, including women of child-bearing potential or pregnant. Nonetheless, neuropsychiatric ...symptoms are frequently reported. Early reports suggested that, probably in relation to folic acid (FA) shortage, DTG may induce neural tube defects in infants born to women taking the drug during pregnancy. Subsequent reports did not definitively confirm these findings. Recent studies in animal models have highlighted the association between DTG exposure in utero and congenital anomalies, and an increased risk of neurologic abnormalities in children exposed during in utero life has been reported. Underlying mechanisms for DTG-related neurologic symptoms and congenital anomalies are not fully understood. We aimed to deepen our knowledge on the neurodevelopmental effects of DTG exposure and further explore the protective role of FA by the use of zebrafish embryos. We treated embryos at 4 and up to 144 h post fertilization (hpf) with a subtherapeutic DTG concentration (1 μM) and observed the disruption of the anterior-posterior axis and several morphological malformations in the developing brain that were both prevented by pre-exposure (2 hpf) and rescued by post-exposure (10 hpf) with FA. By whole-mount in situ hybridization with riboprobes for genes that are crucial during the early phases of neurodevelopment (
,
,
,
) and by in vivo visualization of the transgenic Tg(
:EGFP) zebrafish line, we found that DTG induced severe neurodevelopmental defects over time in most regions of the nervous system (notochord, midbrain-hindbrain boundary, eye, forebrain, midbrain, hindbrain, spinal cord) that were mostly but not completely rescued by FA supplementation. Of note, we observed the disruption of
expression in the dopaminergic regions of the developing forebrain, spinal cord neurons and spinal motor neuron projections, with the depletion of the tyrosine hydroxylase (TH)
dopaminergic neurons of the dorsal diencephalon and the strong reduction in larvae locomotion. Our study further supports previous evidence that DTG can interfere with FA pathways in the developing brain but also provides new insights regarding the mechanisms involved in the increased risk of DTG-associated fetal neurodevelopmental defects and adverse neurologic outcomes in in utero exposed children, suggesting the impairment of dopaminergic pathways.
The aquatic ecotoxicity of three α-amino acid-derived polyamidoamines (PAAs) was studied using zebrafish embryos as a viable vertebrate model organism. The PAAs examined were water-soluble amphoteric ...polyelectrolytes with a primarily negative charge, which were efficient flame retardants for cotton. The fish embryo acute toxicity test performed with PAA water solutions using 1.5–500 mg L−1 concentrations showed that toxicity did not statistically differ from the control. The survival rates were indeed >90%, even at the highest concentration; the hatching rates were >80%; and the numbers of morphological defects were comparable to those of the control. Tests using transgenic zebrafish lines indicated that the numbers of microscopic vascular and musculoskeletal defects were comparable to the control, with one random concentration showing doubled alterations. Sensory–motor tests in response to visual and tactile stimuli were also performed. In the presence of PAAs, embryos exposed to alternating light/dark cycles showed an insignificant mobility reduction during the dark phase. Touch-evoked response tests revealed a mild effect of PAAs on the neuromotor system at concentrations > 10 mg L−1. The cystine/glycine copolymer at 100 mg L−1 exhibited the greatest effect. Overall, the studied PAAs showed a minimal impact on aquatic systems and should be further considered as promising ecofriendly materials.
Coenzyme A (CoA) is an essential cofactor in all living organisms. It is involved in a large number of biochemical processes functioning either as an activator of molecules with carbonyl groups or as ...a carrier of acyl moieties. Together with its thioester derivatives, it plays a central role in cell metabolism, post-translational modification, and gene expression. Furthermore, recent studies revealed a role for CoA in the redox regulation by the S-thiolation of cysteine residues in cellular proteins. The intracellular concentration and distribution in different cellular compartments of CoA and its derivatives are controlled by several extracellular stimuli such as nutrients, hormones, metabolites, and cellular stresses. Perturbations of the biosynthesis and homeostasis of CoA and/or acyl-CoA are connected with several pathological conditions, including cancer, myopathies, and cardiomyopathies. In the most recent years, defects in genes involved in CoA production and distribution have been found in patients affected by rare forms of neurodegenerative and neurodevelopmental disorders. In this review, we will summarize the most relevant aspects of CoA cellular metabolism, their role in the pathogenesis of selected neurodevelopmental and neurodegenerative disorders, and recent advancements in the search for therapeutic approaches for such diseases.
In the past, one of the most widely used non-nucleoside reverse transcriptase inhibitors (NNRTI) in first-line antiretroviral therapy (ART) of HIV infection was efavirenz (EFV), which is already used ...as a cost-effective treatment in developing countries due to its efficacy, tolerability, and availability. However, EFV also demonstrates several adverse effects, like hepatotoxicity, altered lipid profile, neuropsychological symptoms, and behavioral effects in children after in utero exposure. In 2018, another NNRTI, doravirine (DOR), was approved due to its similar efficacy but better safety profile. Preclinical safety studies demonstrated that DOR is not genotoxic and exhibits no developmental toxicity or effects on fertility in rats. Zebrafish (
) embryos have been widely accepted as a vertebrate model for pharmacological and developmental studies. We used zebrafish embryos as an in vivo model to investigate the developmental toxicity of DOR compared to EFV. After exposure of the embryos to the drugs from the gastrula stage up to different developmental stages (30 embryos for each arm, in three independent experiments), we assessed their survival, morphology, hatching rate, apoptosis in the developing head, locomotion behavior, vasculature development, and neutral lipid distribution. Overall, DOR showed a better safety profile than EFV in our model. Therapeutic and supra-therapeutic doses of DOR induced very low mortality survival rates: 92, 90, 88, 88, and 81% at 1, 5, 10, 25, and 50 μM, respectively, at 24 h post fecundation (hpf), and 88, 85, 88, 89, and 75% at the same doses, respectively, at 48 hpf and mild morphological alterations compared to EFV exposure also in the sub-therapeutic ranges (survival rates: 80, 77, 69, 63, and 44% at 1, 5, 10, 25, and 50 μM, respectively, at 24 hpf and 72, 70, 63, 52, and 0% at the same doses, respectively, at 48 hpf). Further, DOR only slightly affected the hatching rate at supra-therapeutic doses (97, 98, 96, 87, and 83% at 1, 5, 10, 25, and 50 μM, respectively, at 72 hpf), while EFV already strongly reduced hatching at sub-therapeutic doses (83, 49, 11, 0, and 0% at 1, 5, 10, 25, and 50 μM, respectively, at the same time endpoint). Both DOR at therapeutic doses and most severely EFV at sub-therapeutic doses enhanced apoptosis in the developing head during crucial phases of embryo neurodevelopment and perturbed the locomotor behavior. Furthermore, EFV strongly affected angiogenesis and disturbed neutral lipid homeostasis even at sub-therapeutic doses compared to DOR at therapeutic concentrations. Our findings in zebrafish embryos add further data confirming the higher safety of DOR with respect to EFV regarding embryo development, neurogenesis, angiogenesis, and lipid metabolism. Further studies are needed to explore the molecular mechanisms underlying the better pharmacological safety profile of DOR, and further human studies are required to confirm these results in the zebrafish animal model.
Sialidases remove terminal sialic acids residues from the non-reducing ends of glycoconjugates. They have been recognized as catabolic enzymes that work within different subcellular compartments and ...can ensure the proper turn-over of glycoconjugates. Four mammalian sialidases (NEU1-4) exist, with different subcellular localization, pH optimum and substrate specificity. In zebrafish, seven different sialidases, with high homology to mammalian counterparts, have been identified. Zebrafish Neu3.2 is similar to the human cytosolic sialidase NEU2, which is involved in skeletal muscle differentiation and exhibits a broad substrate specificity toward gangliosides and glycoproteins. In zebrafish
, mRNA is expressed during somite development, and its enzymatic activity has been detected in the skeletal muscle and heart of adult animals. In this paper, 1-4-cell-stage embryos injected with
splice-blocking morpholino showed severe embryonic defects, mainly in somites, heart and anterior-posterior axis formation.
and
expressions were altered in morphants, and impaired musculature formation was associated with a defective locomotor behavior. Finally, the co-injection of
mouse mRNA in morphants rescued the phenotype. These data are consistent with the involvement of cytosolic sialidase in pathologies related to muscle formation and support the validity of the model to investigate the pathogenesis of the diseases.
Coenzyme A (CoA) is an essential cofactor in all living organisms, being involved in a large number of chemical reactions. Sequence variations in pantothenate kinase 2 (PANK2), the first enzyme of ...CoA biosynthesis, are found in patients affected by Pantothenate Kinase Associated Neurodegeneration (PKAN), one of the most common forms of neurodegeneration, with brain iron accumulation. Knowledge about the biochemical and molecular features of this disorder has increased a lot in recent years. Nonetheless, the main culprit of the pathology is not well defined, and no treatment option is available yet. In order to contribute to the understanding of this disease and facilitate the search for therapies, we explored the potential of the zebrafish animal model and generated lines carrying biallelic mutations in the pank2 gene. The phenotypic characterization of pank2-mutant embryos revealed anomalies in the development of venous vascular structures and germ cells. Adult fish showed testicular atrophy and altered behavioral response in an anxiety test but no evident signs of neurodegeneration. The study suggests that selected cell and tissue types show a higher vulnerability to pank2 deficiency in zebrafish. Deciphering the biological basis of this phenomenon could provide relevant clues for better understanding and treating PKAN.
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