Modeling CLN3 and ATP13A2 deficiency in yeast and zebrafish and use of the ATP13A2 models for drug repurposing

Doctoral thesis (2019)

Neuronal ceroid lipofuscinoses (NCL) are a heterogeneous group of inherited recessive neurodegenerative disorders that appear during childhood and result in premature death. Nowadays, mutations in 14 ... [more ▼]

Neuronal ceroid lipofuscinoses (NCL) are a heterogeneous group of inherited recessive neurodegenerative disorders that appear during childhood and result in premature death. Nowadays, mutations in 14 genes are known to cause NCL and this project focused on CLN3 and ATP13A2 (CLN12), two genes linked to a juvenile form of NCL (JNCL). Mutations in CLN12 are known to cause two additional rare neurodegenerative disorders called Kufor-Rakeb syndrome and spastic paraplegia- 78. Since the number of people affected with a rare disease is relatively small and the cost of the drug development process is high, the chance for a patient to get therapeutic treatment is very low. Therefore, the aim of this PhD project was to develop a new drug screening pipeline for the identification of drug candidates that could be used for the treatment of some of these rare diseases. In this work, we successfully developed a phenotypic high-throughput assay based on a decreased zinc resistance phenotype in an ATP13A2-deficient yeast model and we screened more than 2500 compounds, resulting in the identification of 11 hits. Subsequently, we created a stable ATP13A2 knockout line in zebrafish and developed a validation platform based on decreased manganese resistance in this line. Using this approach, N-acetylcysteine and furaltadone emerged as promising compounds for follow-up studies. A similar strategy could not be implemented for CLN3, due to failure, despite extensive efforts, to find a suitable phenotype in yeast for a drug screening. Nevertheless, we successfully created two stable cln3 mutant lines in zebrafish. No overt phenotype was initially observed, but behavioral tests suggested that cln3 mutants display subtle neurological dysfunction, making them more susceptible to treatment with picrotoxin, a pro-convulsive drug. Further investigation is needed, but our preliminary data indicate that cln3 mutant larvae may recapitulate certain aspects of JNCL pathology. On the whole, this work provides a time- and cost-efficient pipeline for the discovery of drugs against ATP13A2 deficiencies, which can be applied for the screening of larger compound libraries in the future. In addition, we generated a new CLN3 disease model in zebrafish that will be instrumental for the development of drug screens and also may help to elucidate the molecular disease mechanism of JNCL. [less ▲]