Generation of novel developmental and adult zebrafish models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterisedby the death of upper and lower motor neurons. Approximately 10% of ALS patients have a known family history of the disease and genetic analysis of ALS-affected families has identified causal mutations in multiple genes. The identification of these mutations has provided the opportunity to develop models of ALS, essential tools for studies investigating the biology of the disease and for preclinical testing of potential therapeutics. While many of the mechanisms underlying ALS have been elucidated, these mechanisms remain poorly understood. Further studies using both established and novel models of ALS are required to enhance the current understanding of these mechanisms. A greater understanding of disease biology will lead to the identification of potential therapeutic targets. Mutations in CCNF linked to both familial and sporadic ALS were recently reported. Patients who carry these CCNF mutations develop TDP-43 positive protein aggregates within the their motor neurons - pathology considered to be the hallmark of the disease in over 95% of cases. Therefore, the identification of ALS-linked mutations in CCNF provides an opportunity to develop novel models that reflect the most common pathology seen in ALS patients. This project aimed to develop these novel models in the zebrafish. Zebrafish have emerged as useful tools to identify and investigate mechanisms of human disease. As vertebrates, they share significant genetic, anatomical and physiological similarities with humans, while their speed of development, their high fertility and the relative ease of manipulating their genome contribute to efficient development of disease models. This project investigated the suitability of zebrafish to model ALS-linked mutations in CCNF by characterising the zebrafish CCNF homologue and its encoded protein, cyclin F. Comparison of zebrafish and human cyclin F identified significant structural similarities between the proteins, suggesting that they perform similar functions in the two species. Further, cyclin F was found to be persistently expressed in the zebrafish central nervous system throughout development. This suggests that models in which cyclin F is artificially expressed in the central nervous system will have physiological relevance. These findings supported the hypothesis that zebrafish are a suitable species in which to model cellular changes associated with ALS-linked mutant CCNF. Based on these findings, generation of the CCNF-based zebrafish commenced. A variety of model paradigms were explored to identify strategies that produced models suitable for investigative studies. Several strategies failed to generate viable models, including persistent embryonic overexpression of CCNF and selective expression of CCNF within the motor neurons. Two strategies emerged that did produce suitable models with which to study ALS - transient overexpression of ALS-linked mutant CCNF and inducible overexpression of ALS-linked mutant CCNF in adult zebrafish. Evidence presented in this thesis indicates that the transient model will prove useful for efficient analysis of the cellular changes associated with mutant CCNF and will be suitable for use in preclinical trials of potential therapeutics, while the inducible transgenic model will prove useful for longitudinal studies aimed at investigating disease biology in an adult animal. Such studies will contribute to a greater understanding ofthe mechanisms involved in disease onset and progression. The presence of TDP-43 pathology in patients who carry a CCNF mutation suggests that findings from these models will be applicable to wider ALS. A greater understanding of the biology of ALS will lead to the identification of potential therapeutic targets, an essential step in the development of desperately needed effective therapies.