Administration of deoxyribonucleosides or inhibition of their catabolism as a pharmacological approach for mitochondrial DNA depletion syndrome

Abstract

Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is characterized by a reduction in mtDNA copy number and consequent mitochondrial dysfunction in affected tissues. A subgroup of MDS is caused by mutations in genes that disrupt deoxyribonucleotide metabolism, which ultimately leads to limited availability of one or several deoxyribonucleoside triphosphates (dNTPs), and subsequent mtDNA depletion. Here, using in vitro experimental approaches (primary cell culture of deoxyguanosine kinase-deficient cells and thymidine-induced mtDNA depletion in culture as a model of mitochondrial neurogastrointestinal encephalomyopathy, MNGIE), we show that supplements of those deoxyribonucleosides (dNs) involved in each biochemical defect (deoxyguanosine or deoxycytidine, dCtd) prevents mtDNA copy number reduction. Similar effects can be obtained by specific inhibition of dN catabolism using tetrahydrouridine (THU; inhibitor of cytidine deaminase) or immucillin H (inhibitor of purine nucleoside phosphorylase). In addition, using an MNGIE animal model, we provide evidence that mitochondrial dNTP content can be modulated in vivo by systemic administration of dCtd or THU. In spite of the severity associated with diseases due to defects in mtDNA replication, there are currently no effective therapeutic options available. Only in the case of MNGIE, allogeneic hematopoietic stem cell transplantation has proven efficient as a long-term therapeutic strategy. We propose increasing cellular availability of the deficient dNTP precursor by direct administration of the dN or inhibition of its catabolism, as a potential treatment for mtDNA depletion syndrome caused by defects in dNTP metabolism.

Figure 1.

Representation of the main dNTP metabolic pathways. Catabolic enzymes are marked in boxes and inhibitors used in this study are written in gray italics. ADA, adenosine deaminase; CDA, cytidine deaminase; dAdo, deoxyadenosine; dCK, deoxycytidine kinase; dCtd, deoxycytidine; dCTD, deoxycytidylate deaminase; dGK, deoxyguanosine kinase; dGuo, deoxyguanosine; dIno, deoxyinosine; dThd, thymidine; dUrd, deoxyuridine; EHNA, erythro-9-(2-hydroxy-3-nonyl) adenine; ENT1, equilibrative nucleoside transporter 1; IH, Immucillin H; PNP, purine nucleoside phosphorylase; RNR, ribonucleotide reductase; THU, tetrahydrouridine; TK1, thymidine kinase 1; TK2, thymidine kinase 2; TP, thymidine phosphorylase; TS, thymidylate synthase.

Figure 2.

Effect of dCtd and THU administration in vitro and in vivo. (A) Influence of dCtd (1, 5 or 20 µm) and THU (5 µm) treatment on mtDNA copy number in dThd-treated (30 µm) quiescent fibroblasts in culture. Cells were collected at the times indicated and the mtDNA/nDNA ratio was assessed. Results are the mean ± SEM of four different cell lines, plotted as percentages of the ratios obtained for parallel untreated cultures. dCtd and dUrd concentration in plasma (B), and deoxycytidine triphosphate in liver and brain mitochondria (C) of Tymp^−/−/Upp1^−/− mice after 3 h of treatment with THU (100 mg/kg), dCtd (400 mg/kg) or THU + dCtd. Boxplots represent the median (horizontal line), interquartile range (box) and minimum and maximum (whiskers), except outliers (full circle) and extreme values (open circles). P-values obtained with the Mann–Whitney U-test: *P < 0.05; **P < 0.01, compared to KO untreated.

Figure 3.

Effect of purine dNs and monophosphates on mtDNA of dGK-deficient cultured cells. (A) Progression of mtDNA copy number in response to dAdo/dGuo treatment in quiescent dGK-deficient fibroblasts. Results are expressed as the mean ± SEM of three different cell lines, and plotted as percentages of the ratios obtained for parallel untreated cultures from healthy controls. (B) Progression of mtDNA copy number in dGK-deficient fibroblasts after treatment with dAdo/dGuo/dAMP/dGMP. Results are expressed as percentages of the ratios obtained for parallel untreated cultures.

Figure 4.

Effect of a combined administration of purine deoxyribonucloeosides and specific inhibitors of their catabolism on mtDNA of dGK-deficient cultured cells. (A) Progression of mtDNA copy number in response to dGuo and IH (1 µm) treatment in quiescent dGK-deficient fibroblasts. Results are expressed as the mean ± SEM of three different cell lines, and plotted as percentages of the ratios obtained for parallel untreated cultures from healthy controls. (B). Effect of dAdo and 5 µm EHNA on mtDNA copy number progression in dGK-deficient fibroblasts. Results are mean values from three-independent cell lines, expressed as percentages of the ratios obtained for parallel untreated cultures. Error bars indicate SEM.