Zebrafish Models of Autosomal Dominant Ataxias.

Hereditary dominant ataxias are a heterogeneous group of neurodegenerative conditions causing cerebellar dysfunction and characterized by progressive motor incoordination. Despite many efforts put into the study of these diseases, there are no effective treatments yet. Zebrafish models are widely used to characterize neuronal disorders due to its conserved vertebrate genetics that easily support genetic edition and their optic transparency that allows observing the intact CNS and its connections. In addition, its small size and external fertilization help to develop high throughput assays of candidate drugs. Here, we discuss the contributions of zebrafish models to the study of dominant ataxias defining phenotypes, genetic function, behavior and possible treatments. In addition, we review the zebrafish models created for X-linked repeat expansion diseases X-fragile/fragile-X tremor ataxia. Most of the models reviewed here presented neuronal damage and locomotor deficits. However, there is a generalized lack of zebrafish adult heterozygous models and there are no knock-in zebrafish models available for these diseases. The models created for dominant ataxias helped to elucidate gene function and mechanisms that cause neuronal damage. In the future, the application of new genetic edition techniques would help to develop more accurate zebrafish models of dominant ataxias

An in vivo drug screen in zebrafish reveals that cyclooxygenase 2-derived prostaglandin D2 promotes spinal cord neurogenesis

The study of neurogenesis is essential to understanding fundamental developmental processes and for the development of cell replacement therapies for central nervous system disorders. Here, we designed an in vivo drug screening protocol in developing zebrafish to find new molecules and signalling pathways regulating neurogenesis in the ventral spinal cord. This unbiased drug screen revealed that 4 cyclooxygenase (COX) inhibitors reduced the generation of serotonergic interneurons in the developing spinal cord. These results fitted very nicely with available single-cell RNAseq data revealing that floor plate cells show differential expression of 1 of the 2 COX2 zebrafish genes (ptgs2a). Indeed, several selective COX2 inhibitors and two different morpholinos against ptgs2a reduced the number of serotonergic neurons in the ventral spinal cord and led to locomotor deficits. Single-cell RNAseq data and different pharmacological manipulations further revealed that COX2-floor plate-derived prostaglandin D2 promotes neurogenesis in the developing spinal cord by promoting mitotic activity in progenitor cells. Rescue experiments using a phosphodiesterase-4 inhibitor suggest that intracellular changes in cAMP levels underlie the effects of COX inhibitors on neurogenesis and locomotion. Our study provides compelling in vivo evidence showing that prostaglandin signalling promotes neurogenesis in the ventral spinal cord.Grant PID2020-115121GB-I00 funded by MCIN/AEI/10.13039/501100011033 to A. Barreiro-Iglesias and L. Sánchez. Grant ED 431C 2021/18 funded by Xunta de Galicia. The European Molecular Biology Organization (EMBO) granted a long-term EMBO fellowship to D. Sobrido-Cameán (ALTF 62-2021).S

Decline in Constitutive Proliferative Activity in the Zebrafish Retina with Ageing

Supplementary Materials The following are available online at https://www.mdpi.com/article/10.3390/ijms222111715/s1. Table S1: Studies demonstrating the presence of proliferating cells in the juvenile/adult retina of different teleost species. File S1: Mean ± S.E.M. data and data on statistical multiple comparisons related to graphs shown in Figure 2; File S2: Mean ± S.E.M. data and data on statistical multiple comparisons related to graphs shown in Figure 3.It is largely assumed that the teleost retina shows continuous and active proliferative and neurogenic activity throughout life. However, when delving into the teleost literature, one finds that assumptions about a highly active and continuous proliferation in the adult retina are based on studies in which proliferation was not quantified in a comparative way at the different life stages or was mainly studied in juveniles/young adults. Here, we performed a systematic and comparative study of the constitutive proliferative activity of the retina from early developing (2 days post-fertilisation) to aged (up to 3–4 years post-fertilisation) zebrafish. The mitotic activity and cell cycle progression were analysed by using immunofluorescence against pH3 and PCNA, respectively. We observed a decline in the cell proliferation in the retina with ageing despite the occurrence of a wave of secondary proliferation during sexual maturation. During this wave of secondary proliferation, the distribution of proliferating and mitotic cells changes from the inner to the outer nuclear layer in the central retina. Importantly, in aged zebrafish, there is a virtual disappearance of mitotic activity. Our results showing a decline in the proliferative activity of the zebrafish retina with ageing are of crucial importance since it is generally assumed that the fish retina has continuous proliferative activity throughout life.This research was funded by Ministerio de Economía Industria y Competitividad (to E.C.), grant number BFU-2017-89861-P; Ministerio de Ciencia e Innovación-Agencia Estatal de Investigación (to A.B.-I.), grant number PID2020-115121GB-I00; Xunta de Galicia (to E.C.), grant number ED431C 2021/18; Xunta de Galicia (to I.H.-N.), grant number ED 481 A2018 216. “The APC was funded by the Xunta de Galicia”. Grants were partially financed by the European Social Fund.S

Developmentally-programmed cellular senescence is conserved and widespread in zebrafish

Cellular senescence is considered a stress response imposing a stable cell cycle arrest to restrict the growth of damaged cells. More recently however, cellular senescence was identified during mouse embryo development at particular structures during specific periods of time. This programmed cell senescence has been proposed to serve developmental and morphogenetic functions and to potentially represent an evolutionary origin of senescence. Cellular senescence has also been described to take place during bird (chick and quail) and amphibian (xenopus and axoltl) development. Fish however, have been described to show a very narrow and restricted pattern of developmental cell senescence. Here we carried out a detailed characterization of senescence during zebrafish development and found it to be conserved and widespread. Apart from yolk and cloaca, previously described structures, we also identified senescence in the developing central nervous system, intestine, liver, pronephric ducts, and crystalline. Interestingly, senescence at these developing structures disappeared upon treatment with senolytic compound ABT-263, supporting their senescent identity and opening the possibility of studying the contribution of this process to development. In summary, our findings extend the description of developmentally-programmed cell senescence to lower vertebrates contributing to the notion of the relevance of this process for embryo developmentFunding at the laboratory of M.C. is provided by the Ministerio de Ciencia, Innovación y Universidades, Fondos Europeos de Desarrollo Regional (FEDER) (RTI2018-095818-B-100). Work in the laboratory of A.B.-I. was funded by grants from the Xunta de Galicia (2016-PG008) and the crowdfunding platform Precipita (FECYT; 2017-CP081). Funding at laboratory of L.S. is provided by Xunta de Galicia (ED431C2018/28)S

Cell senescence contributes to tissue regeneration in zebrafish

Cellular senescence is a stress response that limits the proliferation of damaged cells by establishing a permanent cell cycle arrest. Different stimuli can trigger senescence but excessive production or impaired clearance of these cells can lead to their accumulation during aging with deleterious effects. Despite this potential negative side of cell senescence, its physiological role as a pro‐regenerative and morphogenetic force has emerged recently after the identification of programmed cell senescence during embryogenesis and during wound healing and limb regeneration. Here, we explored the conservation of tissue injury‐induced senescence in a model of complex regeneration, the zebrafish. Fin amputation in adult fish led to the appearance of senescent cells at the site of damage, and their removal impaired tissue regeneration. Despite many conceptual similarities, this tissue repair response is different from developmental senescence. Our results lend support to the notion that cell senescence is a positive response promoting tissue repair and homeostasis.Funding at the laboratory of M.C. is provided by the Ministerio de Ciencia, Innovación y Universidades, Fondos Europeos de Desarrollo Regional (FEDER) (RTI2018‐095818‐B‐100). Work in the laboratory of A.B.‐I. was funded by grants from the Xunta de Galicia (2016‐PG008) and the crowdfunding platform Precipita (FECYT; 2017‐CP081). The laboratory of L.S. is supported by the Regional Government Xunta de Galicia (ED431C 2018/28)S

Zebrafish as a platform to evaluate the potential of lipidic nanoemulsions for gene therapy in cancer

Gene therapy is a promising therapeutic approach that has experienced significant groth in recent decades, with gene nanomedicines reaching the clinics. However, it is still necessary to continue developing novel vectors able to carry, protect, and release the nucleic acids into the target cells, to respond to the widespread demand for new gene therapies to address current unmet clinical needs. We propose here the use of zebrafish embryos as an in vivo platform to evaluate the potential of newly developed nanosystems for gene therapy applications in cancer treatment. Zebrafish embryos have several advantages such as low maintenance costs, transparency, robustness, and a high homology with the human genome. In this work, a new type of putrescine-sphingomyelin nanosystems (PSN), specifically designed for cancer gene therapy applications, was successfully characterized and demonstrated its potential for delivery of plasmid DNA (pDNA) and miRNA (miR). On one hand, we were able to validate a regulatory effect of the PSN/miR on gene expression after injection in embryos of 0 hpf. Additionally, experiments proved the potential of the model to study the transport of the associated nucleic acids (pDNA and miR) upon incubation in zebrafish water. The biodistribution of PSN/pDNA and PSN/miR in vivo was also assessed after microinjection into the zebrafish vasculature, demonstrating that the nucleic acids remained associated with the PSN in an in vivo environment, and could successfully reach disseminated cancer cells in zebrafish xenografts. Altogether, these results demonstrate the potential of zebrafish as an in vivo model to evaluate nanotechnology-based gene therapies for cancer treatment, as well as the capacity of the developed versatile PSN formulation for gene therapy applicationsThis research was funded by the Instituto de Salud Carlos IIIISCIII and the European Regional Development Fund (FEDER) (AC18/00107, AC18/00045, PI18/00176); by the ERA-NET EURONANOMED III project METASTARG (Grant Number JTC 2018-045) and the ERA-NET EURONANOMED III project PANIPAC (Grant Number JTC 2018/041); and by Axencia Galega de Innovación (GAIN), Consellería de Economía, Emprego e Industria (IN607B2021/14). RP was supported by Roche-Chus Joint Unit (IN853B 2018/03) funded by Axencia Galega de Innovación (GAIN), Consellería de Economía, Emprego e Industria. SL, PH, and AP-L. were funded by a Predoctoral fellowship (IN606A-2019/003, IN606A-2018/019 and ED481A-2018/095) from Axencia Galega de Innovación (GAIN, Xunta de Galicia).S

Pluronic®/casein micelles for ophthalmic delivery of resveratrol: In vitro, ex vivo, and in vivo tests

Ocular health may strongly benefit from the supply of antioxidant agents that counteract free radicals and reactive oxygen species responsible for long-term eye diseases. Additionally, natural antioxidants like resveratrol can inhibit bacteria growth and restore natural microbiota. However, their use is hindered by limited solubility, fast degradation, and low ocular permeability. This work aimed to overcome these limitations by preparing single and mixed micelles of Pluronic® F127 and casein that serve as resveratrol nanocarriers. Single and mixed (0.1 % casein) micelles (0.0 to −17.0 mV; 2.4 to 32.7 nm) increased 50-fold resveratrol solubility, remained stable for one month at 4 °C, withstood fast dilution, underwent sol-to-gel transitions in the 23.9–27.1 °C range, and exhibited potent antioxidant properties. All formulations successfully passed the HET-CAM assay but showed Pluronic®-casein dose-dependent toxicity in the zebrafish embryo model. Resveratrol-loaded single and mixed micelles (10–15 mM Pluronic® F127) displayed antimicrobial activity against S. aureus and P. aeruginosa. The micelles favored resveratrol accumulation in cornea and sclera, but mixed micelles showed larger lag times and provided lower amount of resveratrol permeated through sclera. In vivo (rabbit) tests confirmed the safety of resveratrol-loaded single micelles and their capability to supply resveratrol to anterior and posterior eye segments.Funding: The work was supported by MCIN/AEI/10.13039/501100011033 [PID 2020-113881RB-I00 to A.C. and C.A.-L., and PID2020-115121GB-I00 to L.S. and A.B.-I.], Spain, Xunta de Galicia [ED431C 2020/17], and FEDER. M. Vivero-Lopez acknowledges Xunta de Galicia (Consellería de Cultura, Educación e Ordenación Universitaria) for a predoctoral research fellowship [ED481A-2019/120].S

Pluronic®/casein micelles for ophthalmic delivery of resveratrol: in vitro, ex vivo, and in vivo tests

Ocular health may strongly benefit from the supply of antioxidant agents that counteract free radicals and reactive oxygen species responsible for long-term eye diseases. Additionally, natural antioxidants like resveratrol can inhibit bacteria growth and restore natural microbiota. However, their use is hindered by limited solubility, fast degradation, and low ocular permeability. This work aimed to overcome these limitations by preparing single and mixed micelles of Pluronic® F127 and casein that serve as resveratrol nanocarriers. Single and mixed (0.1 % casein) micelles (0.0 to −17.0 mV; 2.4 to 32.7 nm) increased 50-fold resveratrol solubility, remained stable for one month at 4 °C, withstood fast dilution, underwent sol-to-gel transitions in the 23.9–27.1 °C range, and exhibited potent antioxidant properties. All formulations successfully passed the HET-CAM assay but showed Pluronic®-casein dose-dependent toxicity in the zebrafish embryo model. Resveratrol-loaded single and mixed micelles (10–15 mM Pluronic® F127) displayed antimicrobial activity against S. aureus and P. aeruginosa. The micelles favored resveratrol accumulation in cornea and sclera, but mixed micelles showed larger lag times and provided lower amount of resveratrol permeated through sclera. In vivo (rabbit) tests confirmed the safety of resveratrol-loaded single micelles and their capability to supply resveratrol to anterior and posterior eye segmentsThe work was supported by MCIN/AEI/10.13039/501100011033 [PID 2020-113881RB-I00 to A.C. and C.A.-L., and PID2020-115121GB-I00 to L.S. and A.B.-I.], Spain, Xunta de Galicia [ED431C 2020/17], and FEDER. M. Vivero-Lopez acknowledges Xunta de Galicia (Consellería de Cultura, Educación e Ordenación Universitaria) for a predoctoral research fellowship [ED481A-2019/120]S

Pluronic®/casein micelles for ophthalmic delivery of resveratrol: In vitro, ex vivo, and in vivo tests

Received 10 July 2022; Received in revised form 3 October 2022; Accepted 7 October 2022Ocular health may strongly benefit from the supply of antioxidant agents that counteract free radicals and reactive oxygen species responsible for long-term eye diseases. Additionally, natural antioxidants like resveratrol can inhibit bacteria growth and restore natural microbiota. However, their use is hindered by limited solubility, fast degradation, and low ocular permeability. This work aimed to overcome these limitations by preparing single and mixed micelles of Pluronic® F127 and casein that serve as resveratrol nanocarriers. Single and mixed (0.1 % casein) micelles (0.0 to −17.0 mV; 2.4 to 32.7 nm) increased 50-fold resveratrol solubility, remained stable for one month at 4 °C, withstood fast dilution, underwent sol-to-gel transitions in the 23.9–27.1 °C range, and exhibited potent antioxidant properties. All formulations successfully passed the HET-CAM assay but showed Pluronic®-casein dose-dependent toxicity in the zebrafish embryo model. Resveratrol-loaded single and mixed micelles (10–15 mM Pluronic® F127) displayed antimicrobial activity against S. aureus and P. aeruginosa. The micelles favored resveratrol accumulation in cornea and sclera, but mixed micelles showed larger lag times and provided lower amount of resveratrol permeated through sclera. In vivo (rabbit) tests confirmed the safety of resveratrol-loaded single micelles and their capability to supply resveratrol to anterior and posterior eye segments.Depto. de Optometría y VisiónFac. de Óptica y OptometríaTRUEFondos FEDER - European Union’s Horizon 2020Ministerio de Ciencia e Innovación de España - MCIN/AEI/10.13039/ 501100011033Xunta de Galicia (España)pu

Desarrollo de modelos de pez cebra (Danio rerio) para la investigación de SCA36 y otras ataxias

En este proyecto se estudió la contribución de los modelos de pez cebra (Danio rerio) para el estudio de los mecanismos moleculares de las ataxias. Esto permitió analizar las ventajas y limitaciones de estos modelos y decidir que líneas de pez cebra se iban a caracterizar para la presente tesis. Por ello, se caracterizó un modelo de pez cebra de pérdida de función del gen nop56, gen responsable de la ataxia da costa da morte (SCA36) y se crearon modelos de pez cebra de Niemann Pick tipo C, otra ataxia neurometabólica recesiva utilizando la técnica CRISPR/Cas9. Estas líneas se sometieron a análisis fenotípicos, moleculares, genéticos y de comportamiento entre otros, con la finalidad de incrementar el conocimiento existente sobre estas patologías