• Título:  DECIPHERING AUTISM SPECTRUM DISORDER GENOMIC VARIATION THROUGH THE CHARACTERIZATION OF ZEBRAFISH AND HUMAN STEM CELL-BASED MODELS.
• Autor:  Veiga Rúa, Sara
• Universidad:  Universidad de Santiago de Compostela
• Departamento:  Escuela de Doctorado Internacional (EDIUS)
• Fecha de Lectura:  22/04/2024
• Mención Europeo / Mención Internacional: concedido
• Programa de doctorado:  Programa de Doctorado en Medicina Molecular por la Universidad de Santiago de Compostela
• Dirección: 
  • Sánchez Piñón, Laura (CODIRECTOR)
  • Allegue Toscano, Catarina (CODIRECTOR)
• Tribunal: 
  • PÉREZ GONZALEZ, BELEN (PRESIDENTE)
  • Barreiro Iglesias, Antón (SECRETARIO)
  • Crowe, James (VOCAL)
• Descriptores: 
  • GENETICA DEL DESARROLLO
  • NEUROBIOLOGIA MOLECULAR
  • INGENIERIA GENETICA
• Localización:  BIBLIOTECA XERAL USC
• Resumen:
  Autism Spectrum Disorders (ASD) are characterized by social and communicational impairments, in addition to restricted patterns of behaviour. Despite advances towards a better understanding of ASDs neurobiology, the mechanisms underlying these disorders are poorly understood. For this reason, in this thesis in vitro and in vivo models were generated to study the implication of candidate ASD-risk genes in the development of the disorder. To do so, human induced pluripotent stem cells (hiPSCs) were selected as in vitro models. These are undifferentiated cells capable of self-renewal and differentiation into specialized cell types within an organism, including neurons and glia. Combining hiPSCs with the CRISPR-Cas9 system makes it possible to introduce specific genomic variants to replicate or correct point mutations associated with human diseases or disorders. Despite their advantages, cellular models have limited complexity and cannot replicate the core symptoms of ASD. For this reason, zebrafish (Danio rerio) was also proposed as an in vivo model to address the aforementioned limitations. First, an in vitro model was generated and characterized to study BTBD8s role in ASD. BTBD8 encodes a protein with a BTB/POZ domain, potentially involved in transcriptional regulation and synaptic transmission, although the information regarding its role in these processes is limited. Here, CRISPR-Cas9 was employed to knockout the BTBD8 in hiPSCs. As a result, a homogeneous cell population with biallelic mutations in this gene was isolated and characterized. Then, the edited cell line was differentiated into cortical neurons and brain organoids to study the impact of BTBD8 dysfunction on neurodevelopment, which was analysed through immunostaining and proteomic approaches. Results obtained in this thesis support the functional involvement of BTBD8 in the complex network of processes triggering alterations in neurodevelopment, including imbalances in neural progenitor cell proliferation and differentiation, as well as alterations in clathrin-mediated endocytosis, neurotransmitter exocytosis, vesicle transport, and synaptogenesis. Second, FOSL2 role in neurodevelopment was analysed. FOSL2 belongs to a family of transcription factors involved in the regulation of cell proliferation, migration, survival, and differentiation in response to environmental signals. Recently, it was described a direct association between mutations in this gene and the presence of a syndromic developmental disorder with ASD. Here, a fosl2 knockdown model was generated in zebrafish to study gene function during development. The obtained results indicate that fosl2 expression is essential in early development stages in zebrafish. Third, in vitro and in vivo models were generated for the study of the TAOK1 function. TAOK1 is a Ser/Thr kinase involved in the regulation of cellular signalling pathways. Here, an in vitro knockout model was generated in hiPSCs using the CRISPR-Cas9. The edited cell line was differentiated into human brain organoids, characterized through immunostaining and single-cell RNA sequencing (scRNA-seq). As a result, alterations associated with TAOK1 dysfunction were observed in the proliferation of neural progenitor cells, neuronal migration, and neural differentiation. Overall, these data support the crucial regulatory role of TAOK1 in the development of the human nervous system. A mutant zebrafish model was also generated to study the consequences in vivo of blocking the expression of taok1. Morphological and behavioural analyses of this model did not reveal significant differences between mutant individuals and controls. Nevertheless, it is necessary to extend the temporal scale of the study to determine if the lack of expression of the gene of interest causes morphological and locomotor alterations in later stages not covered in this thesis. In summary, the generated models facilitate a deeper understanding of the molecular mechanisms guiding neurodevelopment and its alterations, thus leading to an improved diagnosis offered to patients and their families. In addition, these models open new avenues for the application of innovative therapeutical strategies capable of reversing or alleviating the patient's distinct symptoms. Consequently, this approach signifies a paradigm shift towards personalized and precision diagnosis and therapy, a concept that was scarcely imaginable until just a few years ago.