Limited dCTP availability accounts for mitochondrial DNA depletion in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)

Emiliano González-Vioque, Javier Torres-Torronteras, Antoni L Andreu, Ramon Martí, Emiliano González-Vioque, Javier Torres-Torronteras, Antoni L Andreu, Ramon Martí

Abstract

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a severe human disease caused by mutations in TYMP, the gene encoding thymidine phosphorylase (TP). It belongs to a broader group of disorders characterized by a pronounced reduction in mitochondrial DNA (mtDNA) copy number in one or more tissues. In most cases, these disorders are caused by mutations in genes involved in deoxyribonucleoside triphosphate (dNTP) metabolism. It is generally accepted that imbalances in mitochondrial dNTP pools resulting from these mutations interfere with mtDNA replication. Nonetheless, the precise mechanistic details of this effect, in particular, how an excess of a given dNTP (e.g., imbalanced dTTP excess observed in TP deficiency) might lead to mtDNA depletion, remain largely unclear. Using an in organello replication experimental model with isolated murine liver mitochondria, we observed that overloads of dATP, dGTP, or dCTP did not reduce the mtDNA replication rate. In contrast, an excess of dTTP decreased mtDNA synthesis, but this effect was due to secondary dCTP depletion rather than to the dTTP excess in itself. This was confirmed in human cultured cells, demonstrating that our conclusions do not depend on the experimental model. Our results demonstrate that the mtDNA replication rate is unaffected by an excess of any of the 4 separate dNTPs and is limited by the availability of the dNTP present at the lowest concentration. Therefore, the availability of dNTP is the key factor that leads to mtDNA depletion rather than dNTP imbalances. These results provide the first test of the mechanism that accounts for mtDNA depletion in MNGIE and provide evidence that limited dNTP availability is the common cause of mtDNA depletion due to impaired anabolic or catabolic dNTP pathways. Thus, therapy approaches focusing on restoring the deficient substrates should be explored.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. Schematic representation of the metabolic…
Figure 1. Schematic representation of the metabolic pathways that supply dNTPs for mtDNA replication.
dThd: thymidine; dCtd: deoxycytidine; dAdo: deoxyadenosisne; dGuo: deoxyguanosine; TP: thymidine phosphorylase; TK1 (cytosolic) and TK2 (mitochondrial) thymidine kinases; dGK: deoxyguanosine kinase; dCK: deoxycytidine kinase; cdN and mdN: cytosolic and mitochondrial deoxynucleotidases; dCTD: dCMP deaminase; TS thymidylate synthase; RNR: ribonucleotide reductase. ENT1: equilibrative nucleoside transporter 1, which has been found in human mitochondria , . The circle with a question mark indicates evidence of a highly concentrative dTMP transport by an unidentified carrier. A dCTP transport activity has also been reported . Enzymes involved in mtDNA depletion syndrome are highlighted in grey.
Figure 2. dTTP excess causes a decrease…
Figure 2. dTTP excess causes a decrease of in organello mtDNA synthesis associated with secondary dCTP depletion.
Concentrations (µM) of dNTPs added to the reaction are indicated in the tables attached to the panels. Dashes: no dNTP addition. Asterisks: radiolabeled nucleotide. All results (except panel E) were obtained after 2 hours of in organello reaction. Bars represent mean±SD. A. Effect of an excess of each dNTP on mtDNA replication. Tritium-labeled dATP or dGTP were used. The reference result (open bar) is plotted as the mean of all the experiments, equaled to 100%, the error bar indicates the SD as percentage. P values obtained with the Wilcoxon T-test. B. Representation of AccI-digested mouse mtDNA. OH, origin of heavy strand replication. OL, origin of light strand replication. Dotted lines, fragments visualized in the gels in panel C, which show the bands resulting from the AccI-digestion; [α32P]-labeled DNA was extracted, AccI-digested, and electrophoresed. The top (32P) and bottom (EtBr) gels show the radioactive signal and ethidium bromide staining, respectively. The even distribution of radioactivity in the 3 bands (here and Figure 3B) indicates that de novo replication in our model is distributed thorough the entire mtDNA, in contrast to other situations in which the 7S-fragment is preferentially replicated . D. Effect of dTTP excess on mitochondrial dNTP pools. P values obtained with Mann-Whitney U test. E. Effect of dTTP excess on transport of radioactive label from exogenous 1 µM [5,5′-3H]dCTP to mitochondria. Radioactivity of the mitochondrial pellet was measured and apparent pmoles were estimated from the specific radioactivity of dCTP. F. dTTP-induced decrease of mtDNA replication in the absence of exogenous dCTP and dGTP. P values obtained with Wilcoxon T-test.
Figure 3. dTTP- and thymidine-induced decrease in…
Figure 3. dTTP- and thymidine-induced decrease in mtDNA replication is prevented by dCTP supplementation.
Concentrations (µM) of dNTP or nucleosides added to the reaction are indicated in the tables attached to the panels. Dashes: no addition. Asterisks: radiolabeled nucleotide. All results were obtained after 2 hours of in organello reaction. Bars represent mean±SD. dThd: thymidine; dCtd: deoxycytidine. A, B. mtDNA synthesis in the presence of increasing amounts of dTTP and dCTP; 3H-labeled DNA was quantified in (A) or 32P-labeled DNA was AccI-digested and resolved in (B). C. Supplementation with dCTP excess (but not dATP or dGTP excess) prevented dTTP-induced decrease in mtDNA replication. D. Effect of thymidine excess on mtDNA replication. P values obtained with Wilcoxon T-test. E. Effect of thymidine excess on mitochondrial dNTP pools. P values obtained with Mann-Whitney U test. F. Thymidine-induced decrease of mtDNA replication was prevented by supplementation with excess of deoxycytidine or dCTP. P values obtained with Wilcoxon T-test.
Figure 4. Hypothetical mechanism accounting for decrease…
Figure 4. Hypothetical mechanism accounting for decrease in mtDNA replication caused by thymidine overload.
TK2-catalyzed deoxycytidine phosphorylation is inhibited competitively by thymidine and noncompetitively by dTTP , leading to depletion of dCTP, which becomes the limiting dNTP for mtDNA replication. ENT1: equilibrative nucleoside transporter 1, which has been found in human mitochondria , . The circle with a question mark indicates evidence of a highly concentrative dTMP transport by an unidentified carrier. mdN and cdN: mitochondrial and cytosolic deoxynucleotidases. dThd: thymidine; dCtd: deoxycytidine.
Figure 5. Thymidine-induced mtDNA depletion is prevented…
Figure 5. Thymidine-induced mtDNA depletion is prevented by deoxycytidine supplementation in human cultured cells.
Influence of thymidine and deoxycytidine supplementation on mtDNA copy number in quiescent primary human skin fibroblasts in culture. Cells were collected at the times indicated and mtDNA/nDNA ratio was assessed. Results are plotted as percentages of the ratios obtained for parallel cultures with no nucleoside addition. Error bars represent standard error of the mean of N independent experiments (N = 4 for squares and rhombs; N = 3 for circles and triangles). dThd: thymidine; dCtd: deoxycytidine.

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