biosensing-motor-neuron-membrane-potential-in-live-zebrafish-embryos

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Università degli Studi di Milano

The pathogenic role of SOD1 mutations in amyotrophic lateral sclerosis (ALS) was investigated using a zebrafish disease model stably expressing the ALS-linked G93R mutation. In addition to the main pathological features of ALS shown by adult fish, we found remarkably precocious alterations in the development of motor nerve circuitry and embryo behavior, and suggest that these alterations are prompted by interneuron and motor neuron hyperexcitability triggered by anomalies in the persistent pacemaker sodium current INaP. The riluzole-induced modulation of INaP reduced spinal neuron excitability, reverted the behavioral phenotypes and improved the deficits in motor nerve circuitry development, thus shedding new light on the use of riluzole in the management of ALS. Our findings provide a valid phenotype-based tool for unbiased in vivo drug screening that can be used to develop new therapies.

INaP selective inhibition reverts precocious inter- and motorneurons hyperexcitability in the Sod1-G93R zebrafish ALS model.

Defects in the control of Wnt signaling have emerged as a recurrent mechanism involved in cancer pathogenesis and acute myeloid leukaemia (AML), including the hematopoietic regeneration-associated WNT10B in AC133(bright) leukaemia cells, although the existence of a specific mechanism remains unproven. We have obtained evidences for a recurrent rearrangement, which involved the WNT10B locus (WNT10B(R)) within intron 1 (IVS1) and flanked at the 5' by non-human sequences whose origin remains to be elucidated; it also expressed a transcript variant (WNT10B(IVS1)) which was mainly detected in a cohort of patients with intermediate/unfavorable risk AML. We also identified in two separate cases, affected by AML and breast cancer respectively, a genomic transposable short form of human WNT10B (ht-WNT10B). The intronless ht-WNT10B resembles a long non-coding RNA (lncRNA), which suggests its involvement in a non-random microhomology-mediated recombination generating the rearranged WNT10B(R). Furthermore, our studies supports an autocrine activation primed by the formation of WNT10B-FZD4/5 complexes in the breast cancer MCF7 cells that express the WNT10B(IVS1). Chemical interference of WNT-ligands production by the porcupine inhibitor IWP-2 achieved a dose-dependent suppression of the WNT10B-FZD4/5 interactions. These results present the first evidence for a recurrent rearrangement promoted by a mobile ht-WNT10B oncogene, as a relevant mechanism for Wnt involvement in human cancer.

Intronless WNT10B-short variant underlies new recurrent allele-specific rearrangement in acute myeloid leukaemia.

Corrigendum: Intronless WNT10B-short variant underlies new recurrent allele-specific rearrangement in acute myeloid leukaemia.

Illicit drugs have been recently identified as a serious environmental problem because of the growing evidence regarding their occurrence in aquatic environment and potential toxicity towards non-target organisms. Among them, cocaine (COC) and its main metabolites, namely benzoylecgonine (BE) and ecgonine methyl ester (EME), are commonly measured in freshwaters worldwide at levels that might cause diverse sub-lethal effects to aquatic organisms. Thus, the present study was aimed at investigating the potential adverse effects induced by the exposure to environmental concentrations (0.04, 0.4, 4 and 40 nM) of COC, BE, and EME on zebrafish (Danio rerio) embryos at 96 h post fertilization. Cytotoxicity was assessed by the Trypan Blue exclusion method, while primary and fixed genetic damages were evaluated by the Single Cell Gel Electrophoresis (SCGE) assay, and the DNA diffusion assay together with the Micronucleus test, respectively. The involvement of oxidative stress in the mechanism of action (MoA) of all tested drugs was assessed by measuring the activity of defense enzymes (SOD, CAT, GPx, and GST) and the expression of their encoding genes. Exposure to COC and both metabolites significantly reduced cell viability, increased DNA fragmentation and promoted the onset of apoptotic cells and micronuclei in zebrafish embryos. Results from oxidative stress-related endpoints and gene expression suggested that the observed genotoxicity may be caused by an overproduction of free radicals that imbalanced the oxidative status of embryos. The integration of biomarker responses into a synthetic index showed that at each tested concentration, BE and EME had a similar toxicity and were both more toxic than COC. Our data confirmed the potential toxicity of environmental concentrations of COC, BE, and EME, suggesting the need of further in-depth studies to shed light on their MoA and long-term toxicity towards non-target aquatic species.

Environmental concentrations of cocaine and its main metabolites modulated antioxidant response and caused cyto-genotoxic effects in zebrafish embryo cells.

Laura Prosperi

Department of Biosciences

Università degli Studi di Milano

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos