ZEBRAFISH (DANIO RERIO) AS A MODEL SYSTEM FOR THE FUNCTIONALVALIDATION OF CANDIDATE GENES FOR HUMAN DISEASES

In recent years, zebrafish (Danio rerio) has become increasingly utilized as a model for studying human gene functions and associated diseases. This has been possible due to the very high conservation of genes and of organs and systems which are fully functional within the very first days of embryonic development. The easy and efficient genetic manipulation, the large number of offspring, and the rapid and external development of the embryos make zebrafish ideal for the generation of disease models and for the conduction of large-scale drug screenings. Thus, zebrafish represents a valuable tool to be used as a support, or in particular cases as a substitute, for other animal models in preclinical research. This PhD project aimed precisely at utilizing zebrafish to generate models of human diseases whose pathogenesis is currently poorly understood and for which effective treatments are not yet available. The main research line focused on the zebrafish model for adenosine deaminase 2 deficiency (DADA2), an auto-inflammatory disease caused by loss-of-function mutations in the ADA2 (also termed CECR1) gene and involving the hematological and vascular compartments. Advances in the comprehension of the molecular mechanisms underlying DADA2 and in the development of effective therapies are hampered by the lack of in-vivo models, due to the lack of an ADA2 orthologue in rodents. We generated a zebrafish DADA2 model with loss-of-function of the cecr1b gene, the functionally conserved ADA2 orthologue. We utilized the CRISPR-Cas9 genome editing approach to target cecr1b and generated a transient mutant model. We combined several techniques, imaging tools, and transgenic lines to characterize in detail the hematological, inflammatory, and vascular defects of cecr1b deficient embryos. We found that they recapitulated the main clinical phenotypes of patients and that they effectively responded to currently used pharmacological therapies. We identified an impairment in adenosine-mediated signaling, which we corrected resulting in a significant improvement in hematological and inflammatory defects. We have therefore developed a valuable zebrafish model for studying the physiological function of ADA2 and the pathogenetic mechanisms of DADA2 and for testing new possible therapeutic strategies. In parallel, we have deepened our knowledge of the possible pathogenesis of this disorder, laying the foundations for new potential correction methods. 6 A further line of this PhD work was dedicated to the generation of a zebrafish model of MicroCephaly Primary Hereditary 17 (MCPH17), a neurodevelopmental disorder characterized by microcephaly and mental retardation. Mutations in the CIT gene have been identified as causative for MCPH17. However, the CIT physiological function and the pathogenesis of MCPH17 are still unclear, and there is currently no curative option. To support and implement the data collected on the murine models and to further investigate the neurodevelopmental mechanisms of MCPH17, which are crucial given the congenital nature of the disorder, we generated a zebrafish model with cita deficiency. We induced cita-loss-of-function through a morpholino-mediated knockdown approach, which resulted in a significant reduction in head size, behavioral alterations, and impairment of neuronal cell populations. In parallel with the characterization of the cita morphants phenotypes, we conducted N-acetylcysteine treatments, which resulted beneficial in treating both the microcephalic condition and the behavioral alterations, as in mice. We demonstrated the potential of the zebrafish in supporting mice models for the comprehension of altered neurodevelopmental processes in MCPH17 and in providing a platform for rapid drug screenings. A further line of research, as yet unfinished, focuses on the study of the pathogenicity of a mutation in a gene of unknown function, named GENE-X in the present thesis. GENE-X was identified as possibly responsible for a complex inflammatory/vascular/metabolic syndrome exhibited by a case report. This section of the PhD project aims to characterize the functional role of GENE-X and the pathogenicity of the mutation identified in the case report using zebrafish as a model system. We have set up and validated a gene-x morpholino-mediated knock-down strategy and started the characterization of the resulting phenotypes. Preliminary analyses, currently extended to the inflammatory and vascular phenotypes only, suggest a significative similarity to the symptoms displayed by the patient under investigation. Once completed the characterization of the model, rescue experiments with the wild-type and mutated GENE-X transcripts will be carried out to evaluate, indirectly, the pathogenicity of the disease and investigate its effects. The last line of research included in this PhD project concerns the generation of a zebrafish model for triadin knock-out syndrome (TKOS). TKOS is a rare, inherited arrhythmogenic cardiac disorder, possibly leading to exercise-induced cardiac arrest in early childhood. Homozygous mutations in the triadin (TRDN) gene, encoding for a 7 muscular protein essential for the structural integrity of the calcium release complex and for the regulation of calcium release during excitation–contraction coupling. However, the precise role of trdn and the mechanism responsible for sudden cardiac events in children suffering from TKOS are not entirely clear, and current anti-arrhythmogenic treatments are often not effective. Hence, there is a need for an in vivo TKOS model to further study the function of TRDN and to perform drug screening to rapidly identify new compounds efficient in restoring proper cardiac function. We identified zebrafish as a possible model system for this objective. We generated a trdn-deficient zebrafish model by exploiting the morpholino-mediated knockdown. Through morphological and functional analyses, we excluded significant alterations in the structure and function of skeletal musculature and the heart, but identified a dysregulation of cardiac rhythm, as occurs in TKOS patients. We have also demonstrated how our model responds effectively to the modulation of heart rhythm by currently used drugs in human therapies, strengthening the potential of our model in the study of TKOS-related mechanisms and in the identification of possible new therapeutic targets.