D'Agostino, Ylenia (0007) Generation and characterization of BDNF knock-out in zebrafish by CRISPR/Cas9 system. [Tesi di dottorato]

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Abstract

BDNF is one of the main neurotrophin (NT) expressed in the brain as well in some peripheral sensory neurons. The important role played by BDNF is demonstrated by its well-established effects on axon growth, synaptic plasticity and, not less important, in processes of learning and memory. Moreover, BDNF is becoming a valid candidate for treatment of neurodegenerative disorders such as epileptic seizures, anxiety and depression or Alzheimer's and Parkinson's diseases. However, unless the huge number of studies performed about its role, some points remain controversial and the whole scenario is still far from be fully delineated. Most of the information we currently have about BDNF function derive from studies in mice but, in this case, the major obstacle is represented by the early postnatal lethality of BDNF-/- and, as consequence, all the results are limited to BDNF+/- animals. The main objective of this PhD thesis was the generation of a BDNF knock-out line in zebrafish through the CRISPR/Cas9 technology and the characterization of the mutant phenotype from both molecular and behavioural points of view. My results show that zebrafish BDNF-/-, unlike from what happens in mouse, survive after birth with undetectable levels of BDNF protein and does not present any mutation in undesirable off-target gene. Behavioural test through larvae monitoring reveals a significantly lower activity, suggestive of reduced exploratory behaviour, while several anomalies such as aberrant pharyngeal arches formation or increasing of apoptotic cells underline the complex role played by BDNF and its implication in a variety of biological processes. Differential transcriptomic analysis between mutant and wild type has been also performed in order to identify downstream target gene regulated by BDNF. The ongoing analysis of the RNA-Seq data will provide new insights on the role played by BDNF during the development of the nervous system. Finally, the zebrafish knock-out line also provides a promising tool in terms of diagnosis and treatment of neurodegenerative diseases in which this important NT is implicated.

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