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See detailPhenotypic assays in yeast and zebrafish reveal drugs that rescue ATP13A2 deficiency
Heins Marroquin, Ursula UL; Jung, Paul UL; Cordero Maldonado, Maria Lorena UL et al

in Brain Communications (2019), 1(1), 2-17

Mutations in ATP13A2 (PARK9) are causally linked to the rare neurodegenerative disorders Kufor-Rakeb syndrome, hereditary spastic paraplegia and neuronal ceroid lipofuscinosis. This suggests that ATP13A2 ... [more ▼]

Mutations in ATP13A2 (PARK9) are causally linked to the rare neurodegenerative disorders Kufor-Rakeb syndrome, hereditary spastic paraplegia and neuronal ceroid lipofuscinosis. This suggests that ATP13A2, a lysosomal cation-transporting ATPase, plays a crucial role in neuronal cells. The heterogeneity of the clinical spectrum of ATP13A2-associated disorders is not yet well understood and currently these diseases remain without effective treatment. Interestingly, ATP13A2 is widely conserved among eukaryotes, and the yeast model for ATP13A2 deficiency was the first to indicate a role in heavy metal homeostasis, which was later confirmed in human cells. Here we show that deletion of YPK9 (the yeast ortholog of ATP13A2) in Saccharomyces cerevisiae leads to growth impairment in the presence of Zn2+, Mn2+, Co2+ and Ni2+, with the strongest phenotype being observed in the presence of zinc. Using the ypk9 mutant, we developed a high-throughput growth rescue screen based on the Zn2+ sensitivity phenotype. Screening of two drug libraries identified 11 compounds that rescued growth. Subsequently, we generated a zebrafish model for ATP13A2 deficiency and found that both partial and complete loss of atp13a2 function led to increased sensitivity to Mn2+. Based on this phenotype, we validated two of the FDA-approved drugs found in the yeast screen to also exert a rescue effect in zebrafish – N-acetylcysteine, a potent antioxidant, and furaltadone, a nitrofuran antibiotic. This study further supports that combining the high-throughput screening capacity of yeast with rapid in vivo drug testing in zebrafish can represent an efficient drug repurposing strategy in the context of rare inherited disorders involving conserved genes. This work also deepens the understanding of the role of ATP13A2 in heavy metal detoxification and provides a new in vivo model for investigating ATP13A2 deficiency. [less ▲]

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See detailModeling CLN3 and ATP13A2 deficiency in yeast and zebrafish and use of the ATP13A2 models for drug repurposing
Heins Marroquin, Ursula UL

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 ▲]

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See detailDeep learning image recognition enables efficient genome editing in zebrafish by automated injections
Cordero Maldonado, Maria Lorena UL; Perathoner, Simon; van der Kolk, Kees-Jan et al

in PLoS ONE (2019)

Detailed reference viewed: 116 (2 UL)