Abstract
Deoxyguanosine kinase (dGK) is an essential rate-limiting component of the mitochondrial purine nucleotide salvage pathway, encoded by the nuclear gene encoding deoxyguanosine kinase (DGUOK). Mutations in DGUOK lead to mitochondrial DNA (mtDNA) depletion typically in the liver and brain, causing a hepatocerebral phenotype. Previous work has shown that in cultured DGUOK patient cells it is possible to rescue mtDNA depletion by increasing substrate amounts for dGK. In this study we developed a mutant dguok zebrafish (Danio rerio) line using CRISPR/Cas9 mediated mutagenesis; dguok−/− fish have significantly reduced mtDNA levels compared with wild-type (wt) fish. When supplemented with only one purine nucleoside (dGuo), mtDNA copy number in both mutant and wt juvenile animals was significantly reduced, contrasting with previous cell culture studies, possibly because of nucleotide pool imbalance. However, in adult dguok−/− fish we detected a significant increase in liver mtDNA copy number when supplemented with both purine nucleosides. This study further supports the idea that nucleoside supplementation has a potential therapeutic benefit in mtDNA depletion syndromes by substrate enhancement of the purine nucleoside salvage pathway and might improve the liver pathology in patients.
Highlights
Mitochondrial DNA depletion syndromes (MDS) are a heterogeneous group of autosomal recessive disorders characterized by a severe reduction in mitochondrial DNA copy number in different tissues without mutations in the mtDNA itself [1]
In this study we developed a mutant dguok zebrafish (Danio rerio) line using CRISPR/Cas9 mediated mutagenesis; dguok−/− fish have significantly reduced mtDNA levels compared with wild-type fish
Previous work from other laboratories suggested that good strategies for restoring mitochondrial deoxyribonucleotide triphosphate (dNTP) pools are either bypassing the enzyme defect [17,19,23] or by increasing substrate availability
Summary
Mitochondrial DNA depletion syndromes (MDS) are a heterogeneous group of autosomal recessive disorders characterized by a severe reduction in mitochondrial DNA (mtDNA) copy number in different tissues without mutations in the mtDNA itself [1]. Reduction of mtDNA leads to the inability to maintain the essential respiratory chain (RC) complexes, resulting in impairment in ATP synthesis, and tissues are unable to function normally [5,6,7]. Changes causing these clinically very heterogeneous diseases are autosomal recessive mutations in at least 15 nuclear genes involved in nuclear-mitochondrial intergenomic signalling pathways. The phenotypes for these disorders can be quite varied from isolated ophthalmoplegia to other tissue-specific or multi-system diseases. MtDNA depletion in these conditions may be associated with multiple mtDNA deletions and point mutations
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