Phenotypic assays in yeast and zebrafish reveal drugs that rescue ATP13A2 deficiency

Abstract

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. In this study, we show that the deletion of YPK9 (the yeast orthologue 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 ypk9Δ mutant, we developed a high-throughput growth rescue screen based on the Zn2+ sensitivity phenotype. Screening of two libraries of Food and Drug Administration-approved drugs 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 confirmed two of the 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.

Flavonoids as Potential Drugs for VPS13-Dependent Rare Neurodegenerative Diseases

“…As SDS is inherently toxic, compounds reducing its toxic properties could also be identified and, thus, the other phenotype—cadmium sensitivity—was subsequently tested (see below). We screened the Prestwick drug repurposing library (Prestwick Chemical) previously used in such screens [ 48 , 50 ], which contains drugs accepted for use in humans. Active compounds were identified after 3–5 days by the observation of growth zones around the filter.…”

CLN3 deficiency leads to neurological and metabolic perturbations during early development

“…Further consolidating our results, we found that GPDs accumulate in a similar manner in MUT2 larvae compared with WT2 larvae, demonstrating that CLN3 function is also invalidated in this line. Interestingly, using our targeted LC-MS/MS method, we did not find accumulation of GPDs in a zebrafish model knocked out for ATP13A2/CLN12 (previously described in reference 35 ), suggesting that the abnormal GPD levels might be specific to CLN3 loss of function and not generalizable to all other NCL genes ( Fig S9B ). We also measured GPDs in human-induced pluripotent stem cell–derived cerebral organoids, a state-of-the-art human model for brain development previously described in references 18 and 36 .…”

CLN3 deficiency leads to neurological and metabolic perturbations during early development

“…9A). Interestingly, using our targeted LC-MS/MS method, we did not find accumulation of GPDs in a zebrafish model knocked out for ATP13A2/CLN12 (previously described in (Heins-Marroquin et al 2019)), suggesting that the abnormal GPD levels might be specific for CLN3 lossof-function and not generalizable to all other NCL genes (Supplementary Fig. 9B).…”

“…during early developmental stages, suggests that GPD metabolism is very tightly connected to CLN3 function. The absence of GPD accumulation in our zebrafish model for ATP13A2/CLN12 deficiency (Heins-Marroquin et al 2019), also leading to neuronal lipofuscinosis (Farias et al 2011; Bras et al 2012; Matsui et al 2013), indicates that these compounds may be specific for certain types of NCLs, a hypothesis that will now need to be tested in different models and non-CLN3 Batten disease patients. The zebrafish model has the potential to fill the gaps between cell culture and mammalian models and accelerate the understanding of early metabolic events that lead to the development of a childhood disease that, as of today, is still invariably lethal.…”

“…Total RNA extraction and cDNA synthesis were performed as described previously (Heins-Marroquin et al 2019). 1 μl cDNA of WT and CRISPR cln3 mutant lines was used as PCR template and amplicons were cloned into the pGEMT vector following the manufacturer's instructions (Promega).…”